EX-VIVO FIBROBLAST TRANSDUCTION IN RABBITS RESULTS IN LONG-TERM (GREATER-THAN-600 DAYS) FACTOR-IX EXPRESSION IN A SMALL PERCENTAGE OF ANIMALS

Delivery of human factor IX to the circulation was analyzed in rabbits by ex vivo fibroblast transduction followed by subcutaneous implantat ion. Kinetic studies of human factor IX in rabbits demonstrated a half -life of approximately 16 hr and a volume distribution of 22%, where i ntraperitoneal and subcutaneous bioavailability was three- to sevenfol d lower than by intravenous administration. Ex vivo retroviral transdu ction of autologous fibroblasts was performed on 15 animals. After sub cutaneous injection of fibroblast-collagen mixtures, the expression of human factor IX in rabbit plasma was followed by ELISA, Of 15 rabbits injected, expression of human factor IX was detected in 2 animals, an d expression was long term (>600 days), One animal had stable levels o f human factor IX, at 20 ng/ml, while the second animal had lower and gradually decreasing levels of human factor IX, There were no gross di fferences in pathology at the injection sites, when comparing animals with human factor IX in plasma and those without. Immunological studie s demonstrated antibody formation in response to injection mixture com ponents (including human factor IX), but again there was no correlatio n with immune response and long-term factor IX production in animals. Tissues at the implantation sites were positive for factor IX DNA by P CR analysis, regardless of whether there was detectable plasma factor IX or not. Small numbers of PCR-positive cells were detected in the in ternal organs of the long term-expressing rabbits while similar tissue s were negative in nonexpressing animals.