title: Systemic lentivirus-mediated delivery of short hairpin RNA targeting calcium release-activated calcium channel 3 as gene therapy for collagen-induced arthritis.
abstract: Immune cells, including T cells, B cells, and osteoclasts, in conjunction with their associated cytokines, have been studied as primary molecular therapeutic targets for the management of rheumatoid arthritis (RA) patients. The increase in cytosolic Ca(2+) levels through the activation of store-operated Ca(2+) release-activated channels (CRACs) is involved in mediating a disparate array of cellular responses by these immune cells. This study was undertaken to investigate the feasibility and efficiency of the regulation of Ca(2+) entry in the treatment of RA. To moderately suppress Ca(2+) entry via CRACs, we gene silenced CRACM3, which was induced by systemic application of specific short hairpin RNAs (shRNAs) using a lentiviral-delivery system, in a murine model of collagen-induced arthritis (CIA). The inflammatory responses were determined by measuring the levels of a panel of cytokines and chemokines in the joints and serum. Ag-specific responses were evaluated by determining the cytokine profile of T cells stimulated with autoantigen. We also analyzed the ability of specific CRACM3-shRNA to regulate mature osteoclast function in CIA mice. The therapeutic effect of lentiviral-delivered CRACM3-shRNA was associated with gene silencing of CRACM3, along with the successful biodistribution of the virus. Extracellular Ca(2+) influx in the splenocytes, thymocytes, and knee joint synovial cells was moderately suppressed. Inflammatory responses and autoimmune responses were reduced by CRACM3 gene silencing. A decrease in mature osteoclast activity also was observed in CRACM3-shRNA-treated CIA mice. These results indicate that regulation of Ca(2+) entry through lentivirus-mediated CRACM3 gene silencing is beneficial in the treatment of RA.
authors.affiliation: Department of Pharmacology, Ehime University Graduate School of Medicine, Shitsugawa, Toon-shi, Ehime 791-0295, Japan; and liussmzk@m.ehime-u.ac.jp.
authors.forename: Shuang
authors.initials: S
authors.lastname: Liu
authors.affiliation: Department of Pharmacology, Ehime University Graduate School of Medicine, Shitsugawa, Toon-shi, Ehime 791-0295, Japan; and Department of Bioscience, Integrated Center for Sciences, Ehime University, Shitsukawa, Toon-shi, Ehime 791-0295, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kiyoi
authors.affiliation: Department of Pharmacology, Ehime University Graduate School of Medicine, Shitsugawa, Toon-shi, Ehime 791-0295, Japan; and.
authors.forename: Erika
authors.initials: E
authors.lastname: Takemasa
authors.affiliation: Department of Pharmacology, Ehime University Graduate School of Medicine, Shitsugawa, Toon-shi, Ehime 791-0295, Japan; and.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Maeyama
mesh_terms: D000818:Animals; D001169:Arthritis, Experimental / Q000628:therapy*; D001172:Arthritis, Rheumatoid / Q000628:therapy*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics*; D020013:Calcium Signaling / Q000235:genetics; D016207:Cytokines / Q000096:biosynthesis / Q000097:blood; D003600:Cytosol / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D007719:Knee Joint / Q000378:metabolism; D016086:Lentivirus; D008297:Male; D051379:Mice; D008811:Mice, Inbred DBA; D010010:Osteoclasts / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D013154:Spleen / Q000166:cytology; D013582:Synovial Fluid / Q000166:cytology; D013601:T-Lymphocytes / Q000276:immunology; D060168:Thymocytes / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D016207:Cytokines; C520432:Orai3 protein, mouse; D034741:RNA, Small Interfering; D002118:Calcium