Ex Vivo Fibroblast Transduction in Rabbits Results in Long-Term (>600 Days) Factor IX Expression in a Small Percentage of Animals

Abstract

Delivery of human factor IX to the circulation was analyzed in rabbits by ex vivo fibroblast transduction followed by subcutaneous implantation. Kinetic studies of human factor IX in rabbits demonstrated a half-life of approximately 16 hr and a volume distribution of 22%, where intraperitoneal and subcutaneous bioavailability was three- to sevenfold lower than by intravenous administration. Ex vivo retroviral transduction of autologous fibroblasts was performed on 15 animals. After subcutaneous 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, and expression was long term (>600 days). One animal had stable levels of 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 differences in pathology at the injection sites, when comparing animals with human factor IX in plasma and those without. Immunological studies demonstrated antibody formation in response to injection mixture components (including human factor IX), but again there was no correlation with immune response and long-term factor IX production in animals. Tissues at the implantation sites were positive for factor IX DNA by PCR analysis, regardless of whether there was detectable plasma factor IX or not. Small numbers of PCR-positive cells were detected in the internal organs of the long term-expressing rabbits while similar tissues were negative in nonexpressing animals.

Overview summary

Chen et al. attempt to repeat experiments that form the basis of a human hemophilia gene therapy trial in China. Fifteen rabbits were subjected to ex vivo autologous fibroblast transplantation followed by subcutaneous implantation. Two of these animals were found to have low levels of factor IX expression in their plasma that persisted long term (>600 days). No correlation could be found between the experimental variables and successful factor IX expression. The reproducibility of this procedure and its potential for human application remain open questions.