PERSISTENT SYSTEMIC PRODUCTION OF HUMAN FACTOR-IX IN MICE BY SKELETALMYOBLAST-MEDIATED GENE-TRANSFER - FEASIBILITY OF REPEAT APPLICATION TO OBTAIN THERAPEUTIC LEVELS

Myoblast-mediated gene transfer and its repeated applications were tes ted for achieving a long-term stable systemic production of human fact or IX (hFIX) at a therapeutic level in SCID mice. Primary skeletal myo blasts were stably transfected with a hFIX expression plasmid vector, pdLMe4 beta AhIXm1, which contains a hFIX minigene under the control o f a p-actin promoter with muscle creatine kinase enhancers, Myotubes d erived from the myoblasts produced 1,750 ng hFIX/10(6) cells/24 hours in culture, hFIX secretion by the myoblasts and thereof derived myotub es were equally efficient, and myotubes were shown to have a sufficien t secretory capacity to handle a substantially elevated production of hFIX. After intramuscular injection of 5, 10, and 20 x 10(6) myoblasts , SCID mice stably produced hFIX into the systemic circulation proport ional to the number of implanted cells, and the expression levels were maintained for at least up to 10 months (end of the experiment). Addi tional cell injections administered to animals that originally receive d 10 x 10(6) cells approximately 2 months later elevated the systemic hFIX levels to an average of 182 +/- 21 ng/mL, a therapeutic level, wh ich persisted for at least 8 months (end of the experiment). These res ults indicate that long-term, stable systemic production of hFIX at th erapeutic levels can be achieved by repeated application of myoblast-m ediated gene transfer. (C) 1997 by The American Society of Hematology.