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A5.15 The validation of disease-inducible promoter constructs for gene therapy in rheumatoid arthritis and osteoarthritis in human THP-1 cells.
  1. Mathijs G A Broeren,
  2. Eline A Vermeij,
  3. Onno J Arntz,
  4. Miranda B Bennink,
  5. Emma Sterken,
  6. Wim B van den Berg,
  7. Fons A J van de Loo
  1. Experimental Rheumatology, Radboud University Medical Centre, Nijmegen

Abstract

Background and Objectives Gene therapy applied in the joints of rheumatoid arthritis (RA) patients can provide a local production of biological drugs to prevent side effects that are encountered when administered systemically. Gene therapy using a promoter (the DNA region of the gene that controls the expression) that is disease-responsive can even confine the expression of the biological drug to episodes of disease flares. Disease-inducible gene therapy has already been successful in mouse models of RA (van de Loo et al. 2004). Recently, gene therapy was also applied successfully in a mouse model of osteoarthritis (OA) (Ruan et al. 2013). Our goal is to validate promoters that can be successful in human gene therapy for RA and OA.

Materials and Methods To identify genes with potential for human gene therapy, we analysed microarrays of synovial samples from 20 RA patients, 12 OA patients and 7 patients with no joint disease. The upregulation of the RA-associated genes was analysed in an in vitro model of inflammation, by stimulating human THP-1 monocytes with the TLR2 ligand Pam3Cys. Subsequently, the promoter regions of these genes were cloned in a lentiviral construct expressing the firefly luciferase reporter gene. THP-1 cells were transduced with the promoter constructs and stimulated for 6 hours with 100 ng/ml Pam3Cys or 10% serum from 9 RA patients and 4 healthy volunteers.

Results The microarray analysis showed 8 genes that were upregulated at least 4-fold in RA synovium and 7 genes that were upregulated at least 2.5-fold in OA. The increased expression was confirmed by qPCR measurements. 6 out of the 8 RA genes were upregulated in the Pam3Cys-stimulated THP-1 cells.

When THP-1 cells were transduced with the constructs containing the promoter regions of the selected genes expressing the luciferase reporter gene and stimulated with Pam3Cys, CXCL10 promoter activity increased 15.5-fold, the IL-8 promoter 26.5-fold, the PCDHB16 promoter 12-fold and the CXCL9 promoter 2.6-fold. After stimulation with human serum from RA patients or healthy volunteers, the promoters of CXCL9, IL-8, TSG-6, CXCL10 and ADAM28 showed a higher activity with RA patient serum.

Conclusions These results show that promoters of genes that are upregulated in RA and OA synovium are promising for developing disease-inducible gene therapy in human, which has already been shown to be successful in mice.

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