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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.

Gene replacement offers a potential cure for degenerative disorders caused by a single gene deletion or mutation. Diagnoses of monogenic disorders in the fetus enable prenatal gene replacement which may be beneficial from the perspectives of host inflammatory/immune response, efficacy and disease prevention. Purpose: To evaluate the distribution and expression of reporter gene green fluorescent protein (GFP) in the tissues of mice survived up to one month following in utero gene therapy. Methods: Vesicular Stomatitis Virus-G (VSV-G) pseudotyped lentiviral (LV) vector containing GFP was prepared via triple plasmid co-transfection. Time-mated CD-1 mice underwent individual amniotic sac injection with either 1x106 LV particles or saline (controls) on gestational day 16 (term=21d), and were allowed to undergo spontaneous parturition. Pups were sacrificed on postnatal days 0, 7, 21 and 28, and neonatal and maternal tissues were analyzed for GFP transgene (by DNA polymerase chain reaction; PCR), and transgene expression by quantitative reverse transcriptase (QRT) PCR and immunohistochemistry (IHC). Results: We observed selective transduction of neonatal tissues (trachea, lung, liver, heart, kidney, spleen, intestine, skeletal muscle), in pups undergoing in utero transfection with LV-GFP. Maternal tissues did not contain transgene despite exposure during amniotic injection. Although the numbers of pups analyzed at each postnatal time point was small, we observed variable persistence of GFP expression that appeared to be tissue specific (with persistent expression noted in intestine of 4 week old pups). Conclusions: Neonatal tissue transfection occurs in a variety of tissues following amniotic injection with LV-GFP in this murine model of in utero gene therapy. Transgene persistence and expression patterns observed over the first 4 weeks of life may reflect tissue-specific genomic insertion of transgene that favors persistent transcription in select tissues.