Session Type: Abstract Submissions (ACR)
Interleukin- 4 (IL-4), a Th2-cell released immunomodulatory factor, has potent chondroprotective and anti-inflammatory properties able to inhibit the synthesis of IL-1β and TNFα, and to reduce the inflammation-induced mediators of cartilage breakdown (e.g.: NO, PGE2, MMPs). To optimize cartilage transplants for the application in patients with cartilage defects as a result of osteoarthritis (OA) or rheumatoid arthritis (RA) we developed a vector for the transgenic expression of IL-4 in chondrocytes under the “smart” control of the inflammation-activated cyclooxygenase- 2 promoter (pCox-2) which is inactive under non-inflammatory conditions. For effective translation to the clinic we use the horse as naturally occurring model since the disease in this species parallels the human form of OA.
The non-viral transfection of equine chondrocytes was optimized by comparing a range of different reagents as well as by different vector designs using the co-expression of the reporter gene GFP by flow cytometry. For further investigations, we used vectors containing Cox-2 and IL-4 in combination or alone. After transfection cells were stimulated using 100ng/ml Il-1β to mimic inflammation. Induction of Il-4 was validated on mRNA level and protein level by quantitative PCR, ELISA and Western Blot. To confirm the functional activity of transgenic IL-4 as well as simulation of inflammation we additionally analyzed Cox-2, IL-1β, IL-6, IL-8, MMP-3, MMP-9 and MMP-13 on mRNA level by quantitative PCR.
The transfection efficiency was increased up to 44% by delivering a vector without IRES and using Turbofect (Fermentas). A plasmid vector including IL-4 as therapeutic gene under the control of pCox-2 was proven to be functional since it induced the production of high levels of IL-4 in cell culture supernatants and cell lysates. Furthermore, in the same cultures IL-1β IL-1β, IL-6, IL-8, MMP-3, MMP-9 and MMP-13 expression was significantly decreased whenever IL-4 was expressed.
Our study shows the promising impact of new generation gene therapy where the therapeutic transgenic target IL-4 will only be expressed if inflammatory mediators are present. IL-4 demonstrated chondroprotective and anti-catabolic potential in vitro. Translation into clinical practice: cartilage transplants containing autologous cells with the inflammation sensitive plasmid could exert therapeutic effects when placed close to the defective joint area. We are presently focusing on the application of the new plasmids in a 3D-in-vitro-OA-model including viral gene delivery systems. Eventually, in vivo studies and clinical trials are needed to substantiate the promising potential of our therapy concept.
Horizon Pharma, Inc,
M. F. G. Schmidt,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/interleukin-4-as-promising-anti-inflammatory-transgene-for-gene-therapeutic-application-in-joint-diseases/