Summary
Until recently, the only way to attain high efficiency gene transfer into primary mammalian cells was by using viral vectors. However, recent developments in novel receptor/liposome-based transfection systems have made nonviral gene therapy a real possibility. Described here is a novel, high-efficiency, nonviral protocol for delivery of genes into permeabilized primary cultured cells forming a first step toward ex vivo gene therapy for the repair of full thickness articular cartilage defects. To test the feasibility of the method, a plasmid carrying a marker β-galactosidase (β-gal) gene, driven by a strong mammalian promoter, was introduced into primary cells. The system consisted of a cell-receptor specific ligand attached to a poly-cation scaffold. The plasmid DNA attached to the polycation scaffold by ionic charge interactions. The system achieved greater than 70% efficiency by utilizing a three-step method: 1) Primary cells were permeabilized using a mild detergent (lysolecithin); 2) The β-gal plasmid was allowed to associate with a polycation (poly-l-lysine) core covalently linked to a receptor ligand (transferrin) forming the DNA/poly-l-lysine-transferrin complex (DTPLL complex); and 3) Cationic liposomes were introduced to the DTPLL complex. This system has now been used to transfect primary perichondrium cells and chondrocytes. More than 70% of the primary cells were found to be positive for β-gal activity. For in vivo assessment, D,D-L,l-polylactic acid (PLA) scaffolds (3 mm × 3.7 mm) seeded with the transfected primary perichondrial cells were implanted into experimentally created osteochondral defects in rabbit knees. The transformed cells continued to express β-gal, in vivo for the entire test period of 7 days, as determined by the β-gal assay. We have previously demonstrated that adding exogenous transforming growth factor beta 1 (TGF-β1) can enhance the chondrocytic phenotype of perichondrial cells (Amiel, Goomer, and Coutts 1997; Dounchis et al. 1997). These studies were initiated in order to assess the usefulness of transfected perichondrium cells as vehicles for the localized delivery of TGF-β1 into the repair site. To this end, we have developed a TGF-β1 expression vector and shown that cells transfected with this construct overexpress the TGF-β1 specific mRNAs. This system is now poised for the delivery of therapeutic genes into primary cultured cells to repair damaged or dysfunctional tissues.
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Goomer, R.S., Amiel, D. (2000). Nonviral Gene Therapy: Application in the Repair of Osteochondral Articular Defects. In: Huard, J., Fu, F.H. (eds) Gene Therapy and Tissue Engineering in Orthopaedic and Sports Medicine. Methods in Bioengineering. Birkhäuser Boston. https://doi.org/10.1007/978-1-4612-2126-5_6
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DOI: https://doi.org/10.1007/978-1-4612-2126-5_6
Publisher Name: Birkhäuser Boston
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