Orthodontics and Craniofacial Research 2005-08-01

Non-viral-mediated gene therapy approaches for bone repair.

S R Winn, J C Chen, X Gong, S V Bartholomew, S Shreenivas, W Ozaki

Index: Orthod. Craniofac. Res. 8(3) , 183-90, (2005)

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Abstract

Bone repair strategies continue to be developed for alternatives to autografting, allogeneic implants of banked bone, and other bone substitutes. Efforts have included the delivery of potent growth and/or differentiation factors and the use of gene therapy. For bone regeneration, gene therapy is the delivery, uptake and expression of DNA that has been localized to a wound bed. The objective of the current study is to investigate methods to enhance non-viral-mediated means of gene uptake and expression for use in bone regeneration.Several types of DNA-polymer complexes, either applied directly to baby hamster kidney (BHK) cells, or released from a porous, resorbable gene-activated matrix (GAM), were evaluated in vitro for their ability to transfect cells with a circular plasmid DNA construct expressing green fluorescent protein. Complexes included conjugates containing a lipophilic reagent, liposomes, poly-ethyl-oxazoline, and poly-ethyleneimine (PEI). Data were subjected to analysis of variance and Fisher's protected least significant difference for multiple comparisons with significance established at p < 0.05.Transfection efficiencies of the liposome and PEI complexes improved in vitro when released from resorbable GAMs. The lipophilic reagent FuGene 6 demonstrated abundant uptake and expression in the initial 1- and 2-day evaluation periods. In contrast, the DNA-liposome and PEI GAM complexes demonstrated a sustained release, uptake and expression by the BHK cells at the 2-, 4-, and 7-day, and 4- and 7-day evaluation intervals, respectively.GAM technology appears to improve the functional stability and release duration of incorporated DNA-polymer complexes in the present in vitro studies. The ongoing objective of our research is to develop a localized treatment to improve the uptake and expression of plasmid DNA by non-viral-mediated gene therapy.