Preparation of a recombinant adeno-associated viral vector with a mutation of 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 factor Ⅸ (hFⅨR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFⅨR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFⅨR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFⅨR338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFⅨR338A was more than 1.25×10¹² particle/mL, and then, a mammalian cell line, 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 expression of rAAV-hFⅨR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng·(10⁶ 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-hFⅨR338A, which was as high as the expression of rAAV-hFⅨ-wt (2565.76±64.36) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFⅨ-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFⅨR338A is about 2.46 times higher than that of hFⅨ-wt. It was first reported that a mutation of human factor Ⅸ was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFⅨR338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFⅨ.

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

A series of adeno-associated viral vectors containing a mutation of human factor IX (hFIXR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFIXR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFIXR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hF IX R338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFKR338A was more than 1.25×10¹² particle/mL, and then, a mammalian cell line, C2C12 and the factor IX knock-out mice were transfected with the rAAV-hFIXR338Ain vitro andin vivo. The results show that the high-level expression of rAAV-hFIXR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng · (10⁶ cells)⁻¹ · (24 h)⁻¹ in C2C12 cellin 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 · (10⁶ cells)⁻¹ · (24 h)⁻¹ in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFIX-wt)in vitro andin vivo, there is no any difference between two groups, but the clotting activity of hFIXR338A is about 2.46 times higher than that of hFIX-wt. It was first reported that a mutation of human factor IX was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFIX R338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFK.