[Preparation of rAAV2/hFIX and experimentally application to gene therapy for hemophilia B]

To prepare the rAAV2/hFIX and evaluate the efficiency of the preparation on gene therapy of hemophilia B model mice.

The rAAV-2/hFIX was prepared by "one helper virus-one vector cell line" strategy and transfected both BHK-21 and C2C12 cells in vitro. The hFIX antigen level in cell culture supernatant was assayed. The rAAV-2/hFIX was injected into muscles of hemophilia B model mice and assayed the serum hFIX levels, hFIX clotting activity, bleeding time, 5 min bleeding volume.

The hFIX antigen could be detected from 24 h till 120 h after BHK-21 and C2C12 cells were transfected with highest levels at 24 h reaching (51.0 +/- 6.5) ng/10(5) cells and (68.0 +/- 7.2) ng/10(5) cells, respectively. The rAAV2/hFIX injected mice could efficiently express hFIX and peaked at three weeks after injection, then slowly decreased but low level hFIX antigen was still detectable till 10 weeks after injection. There were significant differences between the high, middle and low dose groups of rAAV2/hFIX and the control group (P < 0.01), the plasma FIX clotting activities in the model mice were improved remarkably, bleeding time was greatly shortened and bleeding in 5 min was decreased. The hFIX expression level and FIX clotting activity of the high dose of rAAV2/hFIX group (1.6 x 10(13) v.g./kg) reached about (387.0 +/- 12.5) ng/ml plasma in contrast with the normal levels of (30.0 +/- 5.5)% at the third week after injection. No rAAV2 vector DNA was detected in the organs except for injected muscle tissue.

The rAAV2/hFIX transfected BHK-21 and C2C12 cells could efficiently express hFIX antigen and was of therapeutic effects for the hemophilia B model mice by intramuscularly injection.The results provide the basis for clinical trial of rAAV2 gene therapy for hemophilia B.