Long-Term In Vivo Gene Expression via Delivery of PEI–DNA Condensates from Porous Polymer Scaffolds

Abstract

Nonviral delivery vectors are attractive for gene therapy approaches in tissue engineering, but suffer from low transfection efficiency and short-term gene expression. We hypothesized that the sustained delivery of poly(ethylenimine) (PEI)-condensed DNA from three-dimensional biodegradable scaffolds that encourage cell infiltration could greatly enhance gene expression. To test this hypothesis, a PEI-condensed plasmid encoding β-galactosidase was incorporated into porous poly(lactide-co-glycolide) (PLG) scaffolds, using a gas foaming process. Four conditions were examined: condensed DNA and uncondensed DNA encapsulated into PLG scaffolds, blank scaffolds, and bolus delivery of condensed DNA in combination with implantation of PLG scaffolds. Implantation of scaffolds incorporating condensed β-galactosidase plasmid into the subcutaneous tissue of rats resulted in a high level of gene expression for the entire 15-week duration of the experiment, as exemplified by extensive positive staining for β-galactosidase gene expression observed on the exterior surface and throughout the cross-sections of the explanted scaffolds. No positive staining could be observed for the control conditions either on the exterior surface or in the cross-section at 8- and 15-week time points. In addition, a high percentage (55–60%) of cells within scaffolds incorporating condensed DNA at 15 weeks demonstrated expression of the DNA, confirming the sustained uptake and expression of the encapsulated plasmid DNA. Quantitative analysis of β-galactosidase gene expression revealed that expression levels in scaffolds incorporating condensed DNA were one order of magnitude higher than those of other conditions at the 2- week time point and nearly two orders of magnitude higher than those of the control conditions at the 8- and 15-week time points. This study demonstrated that the sustained delivery of PEI–condensed plasmid DNA from PLG scaffolds led to an in vivo long-term and high level of gene expression, and this system may find application in areas such as bone tissue engineering.