Disease-Regulated Gene Therapy with Anti-Inflammatory Interleukin-10 Under the Control of the CXCL10 Promoter for the Treatment of Rheumatoid Arthritis

Abstract

Disease-inducible promoters for the treatment of rheumatoid arthritis (RA) have the potential to provide regulated expression of therapeutic proteins in arthritic joints. In this study, we set out to identify promoters of human genes that are upregulated during RA and are suitable to drive the expression of relevant amounts of anti-inflammatory interleukin (IL)-10. Microarray analysis of RA synovial biopsies compared with healthy controls yielded a list of 22 genes upregulated during RA. Of these genes, CXCL10 showed the highest induction in lipopolysaccharide-stimulated synovial cells. The CXCL10 promoter was obtained from human cDNA and cloned into a lentiviral vector carrying firefly luciferase to determine the promoter inducibility in primary synovial cells and in THP-1 cells. The promoter activation was strongest 8-12 hr after stimulation with the proinflammatory cytokine tumor necrosis factor (TNF)-α and was reinducible after 96 hr. In addition, the CXCL10 promoter showed a significant response to RA patient serum, compared with sera from healthy individuals. The luciferase gene was replaced with IL-10 to determine the therapeutic properties of the CXCL10p-IL10 lentiviral vector. Primary synovial cells transduced with CXCL10p-IL10 showed a great increase in IL-10 production after stimulation, which reduced the release of proinflammatory cytokines TNF-α and IL-1β. We conclude that the selected proximal promoter of the CXCL10 gene responds to inflammatory mediators present in the serum of patients with RA and that transduction with the lentiviral CXCL10p-IL10 vector reduces inflammatory cytokine production by primary synovial cells from patients with RA. CXCL10 promoter-regulated IL-10 overexpression can thus provide disease-inducible local gene therapy suitable for RA.

Identifying key genes in rheumatoid arthritis by weighted gene co‐expression network analysis

“…To explore the RA‐related genes and provide an in‐depth insight into RA pathogenesis, two recent published gene expression profile datasets GSE55235 and GSE77298 were downloaded from the GEO (Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/geo/) database. The expression data of 10 patients with RA and 10 healthy controls from the first dataset (GSE55235) were used in the present study (the platform is Agilent‐014850 Whole Human Genome Microarray 4 × 44K G4112F).…”

<p>Role of Chemokines and Chemokine Receptors in Rheumatoid Arthritis</p>

“…144 A study by Broeren et al reported that CXCR3 ligand, CXCL10 increases inflammatory mediators which are present in the serum of patients with RA. 145 The CXCL10 promoter-regulated IL-10 overexpression was described to lead to a reduction in inflammatory cytokine production. For that reason, this vector was suggested to provide a possible gene therapy approach for RA.…”

Genetic modification of ER-Hoxb8 osteoclast precursors using CRISPR/Cas9 as a novel way to allow studies on osteoclast biology

“…The following CRISPR guide oligonucleotides were ordered: GFP control, gRNA (forward: 59-CACCGGGGCGAGGAGCTGTTCACCG-39, reverse 59-AAACCGGTGAACAGCTCCTCGCCCC-39), NFATc1 gRNA (forward: 59-CACCGGTAGTTGGACTCGTAGGAGG-39, reverse: 59-AAACCCTCCTACGA-GTCCAACTACC-39), and DC-STAMP gRNA (forward: 59-CACCGGCTCATA-TGAATGACACTAG-39, reverse: 59-AAACCTAGTGTCATTCATATGAGCC-39) (Biolegio, Nijmegen, The Netherlands) and were cloned using the BsMBI overhangs. After sequence verification of the insert, lentivirus was made according to Broeren et al [21]. Virus concentrations were determined using the INNOTEST HIV antigen mAb kit (Diasorin, Saluggia, Verceli, Italy).…”