Abstract
Gene-directed tissue repair offers the clinician, human or veterinary, the chance to enhance cartilage regeneration and repair at a molecular level. Non-viral plasmid vectors have key biosafety advantages over viral vector systems for regenerative therapies due to their episomal integration however, conventional non-viral vectors can suffer from low transfection efficiency. Our objective was to identify and validate in vitro a novel non-viral gene expression vector that could be utilized for ex vivo and in vivo delivery to stromal-derived mesenchymal stem cells (MSCs). Minicircle plasmid DNA vector containing green fluorescent protein (GFP) was generated and transfected into adipose-derived MSCs from three species: canine, equine and rodent and transfection efficiency was determined. Both canine and rat cells showed transfection efficiencies of approximately 40% using minicircle vectors with equine cells exhibiting lower transfection efficiency. A Sox9-expressing minicircle vector was generated and transfected into canine MSCs. Successful transfection of the minicircle-Sox9 vector was confirmed in canine cells by Sox9 immunostaining. This study demonstrate the application and efficacy of a novel non-viral expression vector in canine and equine MSCs. Minicircle vectors have potential use in gene-directed regenerative therapies in non-rodent animal models for treatment of cartilage injury and repair.
Highlights
Joint injury and disease are debilitating in both humans and animals, often responding poorly to medical intervention
Canine and equine mesenchymal stem cells (MSCs) were isolated from adipose tissue and cultured in vitro
In this study we were able to show that minicircle vectors showed good efficacy over traditional vector systems in mesenchymal stem cells isolated from two non-human species, dogs and horses, species which can provide suitable model systems for human clinical trials as well as for veterinary clinical applications
Summary
Joint injury and disease are debilitating in both humans and animals, often responding poorly to medical intervention. Genetic modification of MSCs with the high-mobility-group (HMG) domain transcription factor Sox9 [10,11,12] alone or in combination with Sox and Sox (Sox trio) [13,14] has been trialed as an approach to improve chondrogenic outcomes for MSC-mediated regenerative therapies, with encouraging results These studies have shown an increased synthesis of extracellular matrix proteins associated with cartilage formation and an up-regulation in the expression of cartilage-specific genes such as type 2 collagen (COL2A1) and aggrecan compared to controls, in both human and rodent cell lines [10,11,15]. There are significant biosafety issues to be overcome before this approach can be applied in a clinical setting, not least of which is the use of genetically modified material for transplantation
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