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Nonviral Gene Therapy: Application in the Repair of Osteochondral Articular Defects

  • Randal S. Goomer
  • David Amiel
Part of the Methods in Bioengineering book series (MB)

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.

Keywords

Polylactic Acid Connective Tissue Growth Factor Cationic Liposome Osteochondral Defect Mild Detergent 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Birkhäuser Boston 2000

Authors and Affiliations

  • Randal S. Goomer
  • David Amiel

There are no affiliations available

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