Optimization of a nonviral transfection system to evaluate Cox-2 controlled interleukin-4 expression for osteoarthritis gene therapy in vitro.
BACKGROUND
Gene therapy appears to have the potential for achieving a long-term remedy for osteoarthritis (OA). However, there is a risk of adverse reactions, especially when using cytomegalovirus-controlled expression. To provide a safe application, we focused on the expression of therapeutic cytokines [e.g. interleukin (IL)-4] in a disease-responsive manner by use of the previously cloned Cox-2 promoter as 'genetic switch'. In the present study, we report the functionality of a controlled gene therapeutic system in an equine osteoarthritic cell model.


METHODS
Different nonviral transfection reagents were tested for their efficiency on equine chondrocytes stimulated with equine IL-1β or lipopolysaccharide to create an inflammatory environment. To optimize the transfection, we successfully redesigned the vector by excluding the internal ribosomal entry site (IRES). The functionality of our Cox-2 promoter construct with respect to expressing IL-4 was proven at the mRNA and protein levels and the anti-inflammatory potential of IL-4 was confirmed by analyzing the expression of IL-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-1, MMP-3 and tumor necrosis factor (TNF)-α using a quantitative polymerase chain reaction.


RESULTS
Nonviral transfection reagents yielded transfection rates from 21% to 44% with control vectors with and without IRES, respectively. Stimulation of equine chondrocytes resulted in a 20-fold increase of mRNA expression of IL-1β. Such exogenous stimulation of chondrocytes transfected with pNCox2-IL4 led to an increase of IL-4 mRNA expression, whereas expression of inflammatory mediators decreased. The timely link between these events confirms the anti-inflammatory potential of synthesized IL-4.


CONCLUSIONS
We consider that this approach has significant potential for translation into a useful anti-inflammation therapy. Molecular tools such as the described therapeutic plasmid pave the way for a local-controlled, self-limiting gene therapy.
Institute of Immunology, Department of Veterinary Medicine, Freie Universität Berlin, Germany; Department of Rheumatology and Clinical Immunology, Charité University Hospital, Berlin, Germany; German Rheumatism Research Center, Berlin, Germany; Berlin-Brandenburg School of Regenerative Therapies, Charité University Hospital, Berlin, Germany.
Annemarie
A
Lang
Johannes
J
Neuhaus
Moritz
M
Pfeiffenberger
Erik
E
Schröder
Igor
I
Ponomarev
Yvonne
Y
Weber
Timo
T
Gaber
Michael F G
MF
Schmidt
D000818:Animals; D002478:Cells, Cultured; D019902:Chondrocytes / Q000276:immunology / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000235:genetics*; D015536:Down-Regulation; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D006736:Horses; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D015847:Interleukin-4 / Q000096:biosynthesis* / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D010003:Osteoarthritis / Q000235:genetics / Q000628:therapy*; D011401:Promoter Regions, Genetic; D014162:Transfection
D018836:Inflammation Mediators; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D015847:Interleukin-4; D051546:Cyclooxygenase 2
Cox-2 promoter; IL-4; gene therapy; horse model; nonviral transfection; osteoarthritis