OBJECTIVETo investigate the expression of human coagulation factor IX (hFIX) gene in human umbilical cord blood (CB) CD34+ cells which was transfected with recombinant adeno-associated virus 2 (rAAV-2).METHODThe CD34+ cells were transfected with rAAV-2/hFIX and cultured for 21 days for inducing differentiation into myeloid, erythroid and megakaryocytes, respectively. The expression of hFIX was studied at mRNA, protein and biological activity levels. The cytotoxicity of rAAV-2 to CD34+ cells was evaluated by cell proliferation, cell vitality, CD antigen expressions and CFU yields.RESULTSThe hFIX mRNA was expressed in the cultured cells which was verified by RT-PCR and DNA sequencing. An elevated level of hFIX expression and biological activities were detected with a maximum amount of 14.10 ng/10(6) cell x 24 h. During the period of 21 day culture, the cell vitality, cell proliferation, CD antigen expression and CFU yields between the transfected and un-transfected groups had no difference(P > 0.05).CONCLUSIONThe human CB CD34+ cells are able to produce functional hFIX after transduction by rAAV-2/hFIX. The cell proliferation and differentiation capacities of the host CD34+ cells were not affected by rAAV-2.

01 Sep 2004·Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi

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

Article

Author: Tan, Shu-ping ; Peng, Ming ; Dong, Xiao-yan ; Chen, Fang-ping ; Wu, Xiao-bing ; Xue, Jing-lun ; Peng, Jian-qiang ; Chen, Li ; Yuan, Hong

OBJECTIVETo prepare the rAAV2/hFIX and evaluate the efficiency of the preparation on gene therapy of hemophilia B model mice.METHODSThe 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.RESULTSThe 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.CONCLUSIONThe 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.

01 Sep 2003·Yao xue xue bao = Acta pharmaceutica Sinica

[Quality control of recombinant adeno-associated virus type 2/human blood coagulation factor IX].

Article

Author: Wang, Jun-zhi ; Gao, Kai ; Wu, Xiao-bing ; Rao, Chun-ming

AIMTo establish quality control requirements and methods for recombinant adeno-associated virus(rAAV) type 2/human blood coagulation factor IX (rAAV-2/hFIX).METHODSIdentification of rAAV genome fragments, potential contaminants including wild type AAV(wtAAV) and helper virus, were detected by PCR. Purity of rAAV-2/hFIX was analyzed by cation-exchange HPLC and SDS-PAGE. Virus partical numbers were performed by dot blot assay. hFIX expression was demonstrated by ELISA and potency of hFIX was verified by APTT.RESULTSIdentity of rAAV-2/hFIX was proved. Residues of wtAAV and helper virus were conformed to requirements. Purity of rAAV-2/hFIX were more than 98%. Partical numbers of rAAV-2/hFIX were more than 1.0 x 10(15) VG.L-1. hFIX expression was more than 20.0 micrograms.L-1. hFIX potency was verified by APTT following rAAV-2/hFIX injected to FIX gene knockout mice, potency results conformed to requirements.CONCLUSIONThe methods and requirements had been established for quality control of rAAV-2/hFIX.

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Indication	Highest Phase	Country/Location	Organization	Date
Hemophilia B	Phase 1	CN	Vector Gene Technology Co. Ltd.	-

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