title: 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 beta-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 beta-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 beta-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 beta-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.
authors.affiliation: Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Yen-Chen
authors.initials: YC
authors.lastname: Huang
authors.forename: Kathryn
authors.initials: K
authors.lastname: Riddle
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Rice
authors.forename: David J
authors.initials: DJ
authors.lastname: Mooney
mesh_terms: D000818:Animals; D004247:DNA / Q000008:administration & dosage* / Q000378:metabolism; D005240:Feasibility Studies; D005612:Freeze Drying; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D007700:Kinetics; D019344:Lactic Acid / Q000378:metabolism*; D008297:Male; D010957:Plasmids; D011094:Polyethyleneimine / Q000008:administration & dosage* / Q000378:metabolism; D011100:Polyglycolic Acid / Q000378:metabolism*; D011108:Polymers / Q000008:administration & dosage* / Q000378:metabolism; D016062:Porosity; D051381:Rats; D011917:Rats, Inbred Lew; D023822:Tissue Engineering; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis / Q000378:metabolism
chemical_list: D011108:Polymers; D011100:Polyglycolic Acid; D019344:Lactic Acid; D011094:Polyethyleneimine; D004247:DNA; D001616:beta-Galactosidase