Background:In utero gene therapy is a noveltherapy for monogenic disorders of the fetus. Viral vector-mediated gene transfer risks endogenous viral recombination, and random transgene insertion causing insertional mutagenesis. Enhanced biosafety alternatives include non-viral vectors such as the complex polymer, chitosan. The purpose of this study was toevaluate chitosan-mediated transfection in a murine model of fetal genetherapy.
Methods:
1. Chitosan colloidal suspensions wereprepared, and particle size analysis in amniotic fluid (AF) was performed using a zetasizer.
2. Plasmid enhanced green fluorescent protein (eGFP)-chitosan constructs were prepared and protection from endogenous digestion in AF was analyzed by gel electrophoresis.
3. 0.25x105 HEK293T cells were transfected over 2 hours with 0.6 ?g of chitosan-peGFP in varying proportions of medium and AF. After 48 h, cells were directly imaged by fluorescence microscopy for eGFP expression and fluorescence activated cell sorting (FACS) sorting was done to determine transfection efficiency.
4. Amniotic sacs of time-mated CD-1 mice were injected with 30
?L chitosan-peGFP (12.5 ug DNA) on G17. Following natural birth, pupswere sacrificed and tissues were examined for eGFP DNA and mRNA by DNA PCR, RT-PCR, and fluorescence microscopy.
Results: Chitosan forms aggregates in AF, and although in vitro transfection efficiency is decreased by AF, eGFP-chitosan delivery into AF achieves transfection and transgene expression in lung and intestine of mice after birth.
Conclusions: In utero delivery of eGFP plasmid by chitosan results in postnatal gene expression, and shows promise for non-viral gene transfer in animal models of fetal gene therapy.
P.T.P.Y. is supported by a Child and Family–UBCMD/PhD Studentship Award.
393. Efficient Gene Transfer to Muscle Satellite Cells by In Utero Intravenous Delivery of Adeno-Associated Virus Vector