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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Winn
S R
SR
Department of Surgery, Division of Plastic and Reconstructive Surgery, School of Medicine, Oregon Health and Science University, OR 97239, USA. winns@ohsu.edu
Chen
J C
JC
Gong
X
X
Bartholomew
S V
SV
Shreenivas
S
S
Ozaki
W
W
D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry; D001861:Bone Regeneration / Q000235:genetics*; D002460:Cell Line; D002784:Cholesterol / Q000737:chemistry; D024042:Collagen Type I / Q000737:chemistry; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005786:Gene Expression Regulation / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D008055:Lipids / Q000737:chemistry; D008081:Liposomes; D008164:Luminescent Proteins / Q000235:genetics; D010080:Oxazoles / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D011091:Polyesters / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D014162:Transfection / Q000379:methods
D001672:Biocompatible Materials; D024042:Collagen Type I; D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; C411955:FuGene; D008055:Lipids; D008081:Liposomes; D008164:Luminescent Proteins; D010080:Oxazoles; D011091:Polyesters; D011108:Polymers; D000644:Quaternary Ammonium Compounds; D049452:Green Fluorescent Proteins; C071857:polyethyloxazoline; C033616:poly(lactide); D011094:Polyethyleneimine; D004247:DNA; D002784:Cholesterol; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane