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

Content Provider	SpringerLink
Author	Lu, Huazhong Chen, Li Wang, Xuefeng Lu, Daru Qiu, Xinfang Xue, Jinglun
Copyright Year	2001
Abstract	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 1.25×10$^{12}$ 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 · (10$^{6}$ 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 · (10$^{6}$ 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.
Starting Page	1367
Ending Page	1371
Page Count	5
File Format	PDF
ISSN	10016538
Journal	Chinese Science Bulletin
Volume Number	46
Issue Number	16
e-ISSN	18619541
Language	English
Publisher	Science in China Press
Publisher Date	2001-01-01
Publisher Place	Beijing
Access Restriction	Subscribed
Subject Keyword	factor IX mutation recombinant adeno-associated viral vector packaging cell Science Life Sciences Physics Chemistry/Food Science Earth Sciences Engineering
Content Type	Text
Resource Type	Article

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