Volume 5 Supplement 1
Gene transfer using artificial chromosomes containing a marker gene in primary cells and rat adjuvant arthritis
© The Author(s) 2003
Received: 14 January 2003
Published: 24 February 2003
Background and objective
Most antirheumatic therapies are given systemically, which may result in complications because of the high dosage needed to achieve therapeutic levels in the joints. Gene therapy might provide a more efficient system to deliver therapeutic compounds at the site of inflammation. The artificial chromosome expression system (ACes) is a unique, nonintegrating, nonviral gene-expression system, which functions like a natural chromosome. This technology offers advantages over current expression systems because it allows stable expression of genes producing single or multiple proteins over long periods. We are developing ex vivo gene therapy using artificial chromosomes containing reporter genes (LacZ or RFP) for local delivery of genes in rats with adjuvant arthritis. The aim of this study was to evaluate the transfection efficiency in cell lines or primary cells, including rat skin fibroblasts (RSFs) and fibroblast-like synoviocytes (FLSs). Furthermore, we investigated the feasibility of local delivery of a marker gene to the joints of rats with adjuvant arthritis (AA) by ex vivo gene therapy.
Transfer efficiency and optimal dose of transfection agent were determined using iododeoxyuridine (IdUrd)-incorporated ACes complexed to Superfect (Qiagen) (Cytometry Vol 44:100–105, 2001). Following transfection, ACes were antibody-labeled and cells were analyzed by flow cytometry and fluorescent photomicroscopy. Using optimized transfection conditions and after growing the cells under selection, we expanded the colonies and determined reporter gene expression. Cell lines or primary cells transfected with Aces containing the LacZ gene or control cells were injected into the right ankle joints of rats with adjuvant arthritis on day 12 after adjuvant immunization. Joints were harvested 2 and 7 days later and assayed for beta-gal activity.
The delivery of intact artificial chromosomes was detected within 24 h post transfection. Maximum delivery rates of 27% were observed. Flow cytometry data correlated well with microscopic analysis. After growing cell lines and primary cells under selection, clones expressing LacZ and RFP were identified. Ex vivo transfer of the LacZ gene to the ankle joints of rats with AA resulted in expression of the marker gene in the synovium. In the other organs, LacZ expression was not detected two days after injection of the cells.
The results demonstrate that IdUrd-labelled ACes can be detected 24 h after transfection. Both cell lines and primary cells can be efficiently transfected with ACes and express the transgene. In addition, we show successful local ex vivo gene transfer in a rat AA model using ACes containing a marker gene. This work demonstrates the potential feasibility of treating arthritis and other connective tissue diseases utilizing ACes as nonviral vectors for gene therapy.