In utero gene delivery using chitosan-DNA nanoparticles in mice.

Background In utero gene transfer is a novel therapy for monogenic disorders diagnosed in the fetus. Enhanced biosafety alternatives to viral vectors include non-viral transfer agents such as chitosan. The purpose of this study was to evaluate in vitro and in utero gene transfer of reporter gene (GFP) using chitosan as a transfer vehicle. Materials and Methods In vitro studies: 1. Chitosan colloidal suspensions were prepared, and particle stability in murine amniotic fluid (AF) was determined. 2. Chitosan-reporter gene (EGFP) constructs were prepared and protection from endogenous digestion in AF was measured by gel electrophoresis. 3. Transfection efficiency (by chitosan-EGFP) of HEK293T cells was determined in varying proportions of medium and AF. In utero studies: Amniotic sacs of time-mated CD-1 mice were injected with chitosan-pEGFP (12.5 μg DNA) on G17. Pups and their dams were sacrificed and tissues were examined for transgene presence and expression. Results Chitosan formed stable aggregates in AF. Although AF decreased in vitro transfection efficiency, in vivo transfection by amniotic injection achieved short-term transgene expression in pup lung and intestine. Conclusions In utero delivery of chitosan-EGFP results in postnatal gene expression, and shows promise for non-viral gene transfer in animal models of fetal gene therapy.