Double and triple gene transfer in arthritis

As single gene transfer using virus-encoded inhibitory molecules such as IL-1Ra, IL-10 and sTNFαRP55 resulted in different benefical effects on cartilage destruction in the SCID mouse model for RA, we examined the outcome of double and triple gene transfer in this model and analyzed the molecular effects induced by this approach.

RA synovial fibroblasts were transduced using adenoviral sTNFαRp55 and retroviral IL10- or IL1Ra-encoding MFG vectors. Thereafter, transduced cells were mixed in various double and triple combinations and coimplanted with human cartilage for 60 days in SCID mice. Protein expression of the transduced genes was monitored throughout the experiment. RNA of transduced cells was isolated and expression of proto-oncogenes and signaling molecules before and after gene transfer was analyzed using RNA arbitrarily primed PCR (RAP-PCR), cDNA expression array and real-time PCR.

When compared to single gene transfer, combination of IL-1Ra and IL-10 overexpression showed the strongest additive effect resulting in both decrease of perichondrocytic cartilage degradation and reduction of invasion of fibroblasts into the cartilage. IL-1Ra and IL-10 double gene transfer resulted also in distinct alterations of gene expression of molecules involved in cell activation and metabolism. In contrast, double gene transfer with TNFαRp55 did not improve the beneficial effects of IL-10 or those of IL-1Ra. Moreover, triple gene transfer using these three inhibitory molecules partly antagonized the benficial effects observed in double gene transfer both in the SCID mouse as well as on the molecular level.

In summary, double gene transfer of inhibitory molecules results in additive effects on cartilage protection and expression of genes involved in cartilage degradation in RA, whereas a less beneficial outcome may result following gene transfer of additional genes.