Nonviral Gene Therapy: Application in the Repair of Osteochondral Articular Defects
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.
KeywordsPolylactic Acid Connective Tissue Growth Factor Cationic Liposome Osteochondral Defect Mild Detergent
Unable to display preview. Download preview PDF.
- Amiel, D., Chu, C.R., Sah, R.L., and Coutts, R.D. 1999. Tissue engineering of articular cartilage: perichondrial cells in osteochondral repair. Cells and Materials 8(1):161–74.Google Scholar
- Amiel, D., Goomer, R.S., and Coutts, R.D. 1997. The chondrogenic phenotype of perichondrial cells used in the repair of osteochondral defects is influenced by TGF-β1 (transforming growth factor-β1). SIROT 97 Inter-Ming. 34 Haifa, Israel.Google Scholar
- Brant, W.O., Goomer, R.S., and Amiel, D. 1997. Assessment of liposome-mediated transfectional efficacy of aged human chondroprogenitor cells. Am Fed Med Res 45(1):159A.Google Scholar
- Cooper, M.J. 1996. Noninfectious gene transfer and expression systems for cancer gene therapy. Sem Oncol 23:172–87.Google Scholar
- Fairbarn, L.J., Cross, M.A., and Arrand, J.R. 1994. Patterson Symposium 1993, Gene Therapy Br J Cancer 59:972–5.Google Scholar
- Galera, P., Redini, F., Vivien, D., Bonaventure, J., Penfornis, H., Loyau, G., and Pjuol, J.-P. 1992. Effect of transforming growth betal (TGF-β1) on matrix synthesis by monolayer cultures of rabbit articular chondrocytes during the differentiation process. Exp Cell Res 200:379–92.PubMedCrossRefGoogle Scholar
- Gao, X. and Huang, L. 1995. Potentiation of cationic liposome mediated gene delivery by polycations. Biochem 35:1027–36.Google Scholar
- Mack, K.D., Walzern, R.L., Lehmann-Bruinsma, K., Powell, J.S., and Zeldis, J.B. 1996. Polylysine enhances cationic liposome mediated transfection of hepatoblastoma cell line Hep G2. Biotech Appl Biochem 23:217–20.Google Scholar
- Sokol, D.L. and Gewirtz, A.M. 1996. Gene therapy: basic concepts and recent advances. Crit Rev Euk Gene Expr 6:29–57.Google Scholar
- Tremblay, S. 1997. Immunogenecity: its role in cell transplantation and gene therapy. SIROT 97 Mtng. 65, Haifa, Israel.Google Scholar
- Wakatani, S., Goto, T., Pineda, S.J., Young, R.G., Mansour, J.M., Caplan, A.I., and Goldberg, V. 1994. Mesenchymal cell based repair of large, full thickness defects of articular cartilage. J Bone Joint Surg 76:579–92.Google Scholar
- Wheeler, C.J., Felgner, P.L., Tsai, Y.J., Marshall, J., Sukhu, L., Doh, S.G., Hartikka, J., Nietupski, J., Manthorpe, M., Nichols, M., Plewe, M., Liang, X., Noeman, J., Smith, A., and Cheng, S. 1996. A novel cationic lipid greatly enhances plasmid DNA delivery and expression in mouse lung. Proc Natl Acad Sci USA 93:11454–9.PubMedCrossRefGoogle Scholar