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T. Kezuka, R. Matsuda, Y. Usui, C. Nishiyama, Y. Matsunaga, Y. Okunuki, N. Yamakawa, M. Takeuchi, H. Goto; Suppression for Murine Experimental Autoimmune Optic Neuritis by Gene Therapy With Calcitonin Gene-Related Peptide (CGRP). Invest. Ophthalmol. Vis. Sci. 2010;51(13):5784.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, we have reported that mature dendritic cell (mDC) transfected with CGRP gene plays an immunosuppressive role in development of murine experimental autoimmune uveoretinitis (EAU). In this study, we investigated whether CGRP-transfected DC potentially suppress murine experimental autoimmune optic neuritis (EAON) and experimental autoimmune encephalomyelitis (EAE) as well as EAU.
Bone marrow cells derived from C57BL/6 mice were cultured with GM-CSF, and differentiated CD11+ immature DC were purified. Immature DC were then developed into mDC upon stimulation with LPS. Coding region of CGRP cDNA was amplified by PCR using cDNA synthesized from total RNA of ARPE-19 cell line. Human CGRP cDNA fragment was inserted into pCR3.1-2FL plasmid. mDC were introduced the resulting plasmid, pCR3.1-2FL-hCGRP, or pCR3.1-2FL (mock) as control group by electroporation, and they were cultured with myelin/oligodendrocyte glycoprotein derived peptide 35-55 (MOG35-55) before injection. C57BL/6 mice were immunized with MOG35-55 to develop EAON and EAE, and were injected mDC transfected with pCR3.1-2FL-hCGRP, or pCR3.1-2FL intravenously on day 9. EAE was diagnosed on the clinical symptoms of the disease and delayed hypersensitivity (DH) were measured on day 13. On day 14, mice were sacrificed and assessed the extent of EAON histopathologically. The severity of EAON in each eye was scored by scale from 0 to 3.
Gene transfer efficiency was 70% cell viability and 50% transfection efficiency in suspension cells. DH was markedly suppressed by injection of mDC with CGRP-gene transfer compared to control group. EAON was developed in 89% of control group, which was reduced to 67% in CGRP-gene transfer group. Although all mice of both groups developed EAE, the average clinical score of control group was 3 and that of CGRP-gene transfer group was 1.5.
Gene therapy with mDC introduced CGRP was effective for suppression of EAON and EAE development.
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