Inflammation‐induced transgene expression in genetically engineered equine mesenchymal stem cells

Abstract

The results of the present study demonstrate that on demand transgene expression from the NFκB-responsive promoter using naturally occurring inflammatory cytokines can be induced in undifferentiated and chondrogenically differentiated equine MSCs. Copyright © 2016 John Wiley & Sons, Ltd.

Cytokine‐induced interleukin‐1 receptor antagonist protein expression in genetically engineered equine mesenchymal stem cells for osteoarthritis treatment

“…Plasmid pAlli10‐target1916 harbouring the coding sequence of the equine IL‐1Ra gene (GenBank accession number U92482) under the control of synthetic NF‐κB‐responsive promoter identical to the one used in our previous work was custom‐synthetized by Trenzyme (Konstanz, Germany). To obtain vector plasmid pSEWNFkBIL‐1Ra, a 753‐bp Eco RI‐ Eco RI fragment containing the NF‐κB‐responsive elements, the minimal CMV promoter and the equine IL‐1Ra gene was isolated from pAlli10‐target1916 plasmid and inserted into the lentiviral vector plasmid pHR'SIN‐SEW linearized using Eco RI.…”

“…To obtain vector plasmid pSEWNFkBIL‐1Ra, a 753‐bp Eco RI‐ Eco RI fragment containing the NF‐κB‐responsive elements, the minimal CMV promoter and the equine IL‐1Ra gene was isolated from pAlli10‐target1916 plasmid and inserted into the lentiviral vector plasmid pHR'SIN‐SEW linearized using Eco RI. Recombinant lentivirus particles were produced and the respective titre was estimated as described previously …”

“…MSCs were isolated from bone marrow collected from the iliac crest of a 3‐year old male horse euthanized for reasons not related to the present study. MSCs were cultured as described previously . MSCs were collected in accordance with the ‘Good Scientific Practice.…”

“…Recently, we have shown that equine MSCs can be efficiently transduced with lentiviral vector in which the firefly luciferase reporter gene was driven by an inflammation inducible promoter . The present study aimed to investigate tuneable IL‐1Ra expression in equine MSCs using a composite, NF‐κB‐responsive enhancer/promoter regulated by the pro‐inflammatory cytokines TNFα and IL‐1β.…”

“…Inflammation inducible promoters have shown their great value in the context of tunable gene therapy. Such disease‐ (inflammation) responsive promoters will be ‘switched on’ only when pro‐inflammatory cytokines are present, and will be silent again once the joint flair has been successfully treated . In these cases, stimulation of the regulatory elements to enhance transgene expression from the outside would not be needed as is the need with tetracycline responsive promoter systems .…”