Preparation of rAAV/hFⅨ by HSV/AAV hybrid helper virusand evaluation of its safety

Hemophilia B is a hemorrhagic disease resulting from Factor Ⅸ gene (hFⅨ) mutation as an X-linked recessive inherited trait. The incidence of this disease is 1 in 30000. Clinical treatments depend mainly upon blood transfusions or administration of prothrombin complex so that patients are at the risk of infections with the HIV and hepatitis viruses. Gene therapy offers an attractive alternative in the treatment of hemophilia B by eliminating those risks. In 1991, our lab conducted clinica…

Preparation of rAAV/hFlX by HSV/AAV hybrid helper virus and evaluation of its safety

The recombinant adeno-associated viral vector with human coagulation Factor IX minigene which was regulated by CMV promoter was constructed. Large quantity of recombinant adeno-associated viral particles (rAAV/ hFIX) was prepared by the HSV/AAV hybrid helper virus method. Southern dot blot assay and QC-PCR indicated that the titer of the virus was 3.6X10¹² v.g./mL. It demonstrated that this method can effectively overcome the hurdles of mass production of AAV vector. Followed by an intramuscular injection of viral vectors (7.5X10¹¹ v.g./mouse) in the quadriceps femoris, an elevation of human Factor IX expression in the plasma of hemophilia B mice was detected (387 ng/mL) and persisted more than 12 weeks. The level of anti-virus antibody in plasma aligned with the Factor IX expression curve. The QC-PCR method is easier and more accurate than traditional dot hybridization for determination of the titer of recombinant adeno-associated virus. Moreover, there are no HSV particles existing in produced AAV assayed by RT-PCR. AAV is the only virus that has been amplified from AAV-injected muscle by PCR.