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 The purpose of our study was to evaluate the distribution and expression of reporter gene green fluorescent protein (GFP) in the tissues of mice surviving up to 1 month following in utero gene therapy.
Methods Used Vesicular stomatitis virus-G (VSV-G) pseudotyped lentiviral (LV) vector containing GFP was prepared via triple plasmid cotransfection. Time-mated CD-1 mice underwent individual amniotic sac injection with either 1 × 106 LV particles or saline (controls) on gestational day 16 (term = 21 days) 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).
Summary of 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 number 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 the 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.
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