RETROVIRAL-MEDIATED TRANSFER AND AMPLIFICATION OF A FUNCTIONAL HUMAN FACTOR-VIII GENE
- 1 March 1990
- journal article
- research article
- Vol. 75 (5) , 1074-1080
Abstract
Hemophilia A results from a deficiency in factor VIII (FVIII), a cofactor in the intrinsic pathway of blood coagulation. As an approach toward genetic therapy of this disease, we constructed a retroviral vector encoding human FVIII and a selectable and amplifiable genetic marker, human adenosine deaminase (Ada). A retrovirus packaging line was transfected with this vector and stable transformants were selected for Ada expression. Isolated transformants produced both FVIII activity in the conditioned medium and retrovirus capable of transferring the Ada selectable marker and FVIII expression to mouse 3T3 fibroblasts. Selection of virus-producer cell lines for increasing levels of Ada expression yielded a 20-fold increase in both FVII expression and viral titer. Similarly, selection of infected 3T3 fibroblasts for Ada gene amplification yield a 20-fold increase in FVIII expression. The results demonstrate the feasibility of retrovirus-mediated transfer of human FVIII, and also the utility of selection for gene amplication to increase retrovirus titers in producer cell lines as well as expression levels in infected cells.This publication has 18 references indexed in Scilit:
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