Background and Objective
Treatment of rheumatoid arthritis (RA) is problematic with current strategies. Relatively high systemic doses are necessary to achieve therapeutic levels of anti-rheumatic drugs in the joints. Gene therapy might provide a more efficient system to deliver therapeutic compounds at the site of inflammation. Artificial Chromosome Expression System (ACes) is a unique non-integrating, non-viral gene expression system, which functions like a natural chromosome. This technology offers advantages over current expression systems because it allows stable and predictable expression of genes producing single or multiple proteins over long periods of time. We are developing ex vivo gene therapy using a murine artificial chromosome containing a reporter gene (LacZ) for local delivery of genes in rats with adjuvant arthritis. The aim of this study was to evaluate the transfection efficiency of ACes complexed to two commercially available transfection agents into primary cells, such as, rat skin fibroblasts (RSF).
Transfer efficiency and optimal dose of transfection agents was determined using iododeoxyuridine (IdUrd)-incorporated ACes complexed to LipofectAMINE PLUS (Life Technologies) and Superfect (Qiagen) (Cytometry Vol 44:100–105, 2001). Following transfection, the ACes were antibody labelled and the cells were analyzed by FACs for FITC-fluorescence and microscopic staining. Using optimised transfection conditions, hygromycin resistant colonies were expanded and stable, ACes containing, karyotypes were verified by FISH analysis. In addition, β-galactosidase expression was determined to monitor the expression of the reporter gene in the transfected cells.
The delivery of intact artificial chromosomes was detected within 24 to 48 hours post transfection. Maximum delivery rates of 25% were observed. Flow cytometry data correlated well with microscopic observations. After growing the cells under hygromycinB selection, clones expressing LacZ were identified. Stability of the clones is currently under observation.
These data suggest that artificial chromosomes may have potential in ex vivo gene therapy applications using non-viral delivery techniques. Primary cells can be efficiently transfected with ACes and express the transgene. At present we are investigating local delivery of transfected cells to the joints of rats with adjuvant-induced arthritis.