[Human factor VIII expression in retrovirus vector-transduced bone marrow stromal cells].
OBJECTIVE
To explore the feasibility of bone marrow stromal cells (BMSCs) used for gene therapy of hemophilia A.


METHODS
Murine and rabbit BMSCs were transduced with a recombinant replication-incompetent retrovirus LNC-FVIII delta B, which encloses a B-domain-deleted (760aa-1,639aa) human factor VIII cDNA. An optimized protocol involving temperature declining (32 degrees C) and centrifugation was used. Antigen and coagulant activity of human FVIII in the cell culture medium were measured by ELISA assay and one-stage method, respectively. Semi-quantitative PCR was performed for the transduction efficiencies. Western blot was used for FVIII protein analysis.


RESULTS
Transduction efficiencies were increased 5- to 8-fold by temperature decline and centrifugation, which resulted in high expression and secretion of FVIII in transduced mouse[(556 +/- 80) ng/10(6) cells/24 hr] and rabbit [480 +/- 56) ng/10(6) cells/24 hr] BMSCs. Coagulant activities of human FVIII secreted from transduced murine and rabbit BMSCs were 2.42 U/10(6) cells/24 hr and 1.8 U/10(6) cells/24 hr, respectively. Western blot analysis demonstrated that the B domain-deleted FVIII cDNA was secreted predominantly as heavy and light chain heterodimers that resemble the native forms in human plasma.


CONCLUSIONS
It was demonstrated that the BMSCs system has potential utility in the gene therapy for hemophilia A.
Guo
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X
Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Wang
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H
Chu
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H
D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism*; D002498:Centrifugation; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D006467:Hemophilia A / Q000628:therapy; D006801:Humans; D051379:Mice; D011817:Rabbits; D012190:Retroviridae / Q000235:genetics*; D017154:Stromal Cells / Q000378:metabolism; D013696:Temperature; D014161:Transduction, Genetic / Q000379:methods
D005169:Factor VIII