Combination of cellular imaging and molecular analysis for evaluation of cellular gene therapy of RA
© The Author(s) 2003
Received: 14 January 2003
Published: 24 February 2003
Gene therapy has been developed as a promising tool forthe treatment of autoimmune diseases such as rheumatoid arthritis(RA). In order to achieve the goal of establishing a targeted delivery of potentially immune-modulating gene products to inflamed joints in RA, the concept of adoptive cellular gene therapy was developed in an animal model of RA, collagen-induced arthritis (CIA). Adoptive cellular gene therapy utilizes immune cells with specific homing capacity as “vehicle cells” to deliver therapeutic gene products locally after ex vivo retroviral transduction. For the evaluation of adoptive cellular gene therapy two goals have to be achieved. One is the monitoring of cellular trafficking and homing to areas of inflammation. The other is the analysis of molecular effects in the synovium of inflamed target joints. In order to approach these goals, we have tested two analysis powerful techniques on animal and human specimens. For in vivo, real-time visualization of cellular trafficking, bioluminescence imaging has been developed in animal models. For the analysis of compartment-specific analysis of gene expression in human RA synovium, we established the combination of laser-mediated microdissection and differential display to analyze distinct gene expression profiles of histologically defined areas in RA synovium.
In the CIA model, antigen-specific T-cell hybridomas and dendritic cells (DC) were adoptively transferred into recipient animals after ex-vivo retroviral transduction to express luciferase. Repeated injection with the substrate luciferin and bioluminescence imaging on consecutive days allowed in vivo tracking of the adoptively transferred cells. For development of compartment-specific molecular analysis, cryosections derived from RA synovial tissues were used to obtain cells samples from synovial lining and sublining using a microbeam laser microscope. RNA was isolated and analyzed using nested RAPPCR for differential display fingerprinting. Differentially expressed bands were cut out, PCR products were eluted, cloned and sequenced. Differential expression of identified sequences was confirmed by in situ hybridization and immunohistochemistry.
It could be demonstrated that adoptively transferred T-cell hybridomas and DC homed to and accumulated in inflamed joints of CIA mice. In addition, microdissected RA synovial tissue sections containing about 600 cells were shown to yield sufficient RNA for a stable, reproducible RNA fingerprint. This method allowed us to identify several known and unknown genes as being expressed differentially between the synovial lining and sublining layers, including thrombospondin in the linig, Ciz/cip-1 in the sublining and fibronectin in both layers of RA synovium. All three molecules could be confirmed on the mRNA and protein level.
We tested two novel analysis techniques on animal and human specimens. Bioluminescence imaging allowed in vivo monitoring of the migration pattern of therapeutic “vehicle cells” in adoptive cellular gene therapy of CIA, an animal model of RA. Laser microdissection and subsequent RAP-PCR reliably enabled us to obtain novel insights into the area-dependent differential regulation of gene expression in human RA synovium and distinction between different cell types. In combination, these methods present a powerful tool for theevaluation of cellular gene therapy of RA.