[Preliminary experimental research on gene therapy for hemophilia A].
OBJECTIVE
To accomplish a kind of therapeutic gene for hemophilia A, and observe the expression of human factor VIII (hF VIII) in vivo.


METHODS
Human clotting factor VIII cDNA with B-domain deleted (Delta760aa approximately 1639aa) was inserted into vector pRC/RSV to form pRC/RSV-hF VIII BD, which conjugated with in vivo liposome transfection reagent (DOTAP-Cholesterol) to accomplish a kind of therapeutic gene, pRC/RSV-hF VIII BD-DOTAP-Cholesterol. Mice were injected with pRC/RSV-hF VIII BD-DOTAP-Cholesterol i.m. and sacrificed 48 hours, 10 days, 20 days, 30 days, 40 days and 50 days later, respectively. Tissues such as heart, liver, spleen, lung, kidney and muscle were harvested, the distribution and transcription as well as expression of hF VIII BD cDNA were detected by means of PCR, RT-PCR and immunohistochemistry techniques. In addition, the antigen and antibody of hF VIII in plasma were measured.


RESULTS
There was high expression of hF VIII in plasma and tissues at the 48(th) hour after injection. On day 10, antigen level of hF VIII in plasma reached its peak, 17.55 ng/ml, and gradually reduced later. The antibody of hF VIII in plasma emerged on day 10 after injection, and increased and gradually reached 37.06 U/ml on day 50 after injection. PCR, RT-PCR and immunohistochemistry showed that hF VIII BD cDNA and its transcription as well as expression existed in all kinds of tissues, and lasted longer in spleen, lungs and kidneys than in heart, liver and muscle.


CONCLUSIONS
Therapeutic gene, pRC/RSV-hF VIII BD-DOTAP-Cholesterol, produced by combination of pRC/RSV-hF VIII BD and DOTAP-Cholesterol liposome can express human F VIII successfully in vivo, which lays an experimental foundation for curing hemophilia A by gene-drug in clinic.
Yin
Jun
J
Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Wang
Hongli
H
Hu
Yiqun
Y
Wang
Xuefeng
X
Qu
Bin
B
Chu
Haiyan
H
Duan
Baohua
B
Kang
Wenying
W
Qi
Zhengwu
Z
Wang
Zhenyi
Z
D000818:Animals; D018076:DNA, Complementary; D004195:Disease Models, Animal; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use*; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D006467:Hemophilia A / Q000628:therapy*; D006801:Humans; D008081:Liposomes; D051379:Mice; D008807:Mice, Inbred BALB C; D014018:Tissue Distribution; D014162:Transfection
D018076:DNA, Complementary; D008081:Liposomes; D005169:Factor VIII