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Efficient gene transfer into the mouse lung by fetal intratracheal injection of rAAV2/6.2.

Fetal gene therapy is one of the possible new therapeutic strategies for congenital or perinatal diseases with high mortality or morbidity. We developed a novel delivery strategy to inject directly into the fetal mouse trachea. Intratracheal (i.t.) injection at embryonic day 18 (E18) was more efficient in targeting the fetal lung than conventional intra-amniotic (i.a.) delivery. Viral vectors derived from adeno-associated virus serotype 6.2, with tropism for the airway epithelium and not earlier tested in the fetal mouse lung, were injected into the fetal trachea. Bioluminescence (BL) imaging (BLI) was combined with magnetic resonance (MR) imaging (MRI) for noninvasive and accurate localization of transgene expression in vivo. Histological analysis for β-galactosidase (β-gal) revealed 17.5% of epithelial cells transduced in the conducting airways and 1.5% in the alveolar cells. Stable gene expression was observed up to 1 month after injection. This study demonstrates that direct injection of rAAV2/6.2 in the fetal mouse trachea is superior to i.a. delivery for transducing the lung. Second, as stable gene transfer was detected up to 1 postnatal month, this approach may be useful to evaluate fetal gene therapy for pulmonary diseases such as cystic fibrosis, requiring both substantial numbers of transduced cells as well as prolonged gene expression to obtain a stable phenotypic effect.

Antenatal fetal VEGF therapy to promote pulmonary maturation in a preterm rabbit model

AIM To assess the effects of fetal tracheal administration of VEGF on pulmonary maturation in a preterm rabbit model. METHODS On day 26 (term=31days), fetal rabbits received recombinant rat VEGF (30microg in 70microL normal saline) or placebo (normal saline 70microL) intratracheally, with or without subsequent tracheal occlusion. Non-operated littermates served as internal controls. Fetuses were harvested on day 28 for morphometric study of the lungs or for mechanical ventilation and measurement of lung mechanics. In total, 96 fetuses from 42 does were used, 47 for ventilation and 49 for morphometry. RESULTS In fetuses receiving intratracheal VEGF, an increase in immunoreactivity for Flk-1 was observed throughout the lung parenchyma. Tracheal occlusion (TO) adversely affected pulmonary mechanics as compared to un-occluded controls. That effect is partly reversed by intratracheal VEGF. Intratracheal injection of VEGF without tracheal occlusion improves lung mechanics but no more than what was observed in placebo injected controls. CONCLUSION Antenatal intratracheal VEGF administration was associated with an increase in Flk-1 immunoreactivity. It also improves lung mechanics, however more so when the trachea is occluded. Without TO, the effects were comparable to placebo controls.

Maternal administration of betamethasone inhibits proliferation induced by fetal tracheal occlusion in the nitrofen rat model for congenital diaphragmatic hernia: a placebo-controlled study

PurposeFetal tracheal occlusion (TO) is offered to fetuses with severe pulmonary hypoplasia due to congenital diaphragmatic hernia (CDH). TO induces lung growth, but even when performed minimally invasive, there is a risk for iatrogenic preterm delivery. Whenever this is anticipated, maternal glucocorticoids (GC) may be given to enhance lung maturation. The pulmonary effects of GC in fetuses with CDH that underwent TO are yet poorly defined. Therefore, we conducted a placebo-controlled study in the nitrofen (NF) rat model for CDH.MethodsPregnant rats were gavage fed NF or olive oil (OO) on ED9.5. At ED19.0, fetuses were either assigned to TO or left untouched. Maternal betamethasone (BM) or saline (PLAC) was administered on ED20. Necropsy was done on ED21.5 to obtain lung-to-body-weight ratio (LBWR), and perform quantitative RT-PCR and fluorescent immunostaining for Ki-67 and proliferating cell nuclear antigen (PCNA) in fetal lungs.ResultsCDH fetuses had a lower LBWR than normal fetuses, but comparable pulmonary PCNA and Ki-67 expression levels. TO increased LBWR, irrespective of maternal BM or PLAC. However, BM but not PLAC inhibited proliferation in TO and unoperated fetuses.ConclusionRats with NF-induced CDH have hypoplastic lungs with normal proliferation indices. TO triggers proliferation, an effect countered by BM.

Albumin as an adjunct to tracheal occlusion in fetal rats with congenital diaphragmatic hernia: a placebo-controlled study

OBJECTIVE We sought to investigate effects of intratracheal albumin injection prior to tracheal occlusion (TO) on lung proliferation in fetal rats with nitrofen-induced congenital diaphragmatic hernia. STUDY DESIGN On embryonic day 19, nitrofen-exposed fetuses underwent TO, TO and 50 microL of either intratracheal albumin 20% or saline, or remained untouched. Main outcome at embryonic day 21.5 was expression of the proliferation marker Ki-67. Secondary outcomes were lung-to-bodyweight ratio (LBWR), tropoelastin expression, density and spatial distribution of elastin, pulmonary/alveolar morphometry, and fetal survival. RESULTS TO increased Ki-67 messenger RNA and LBWR. Albumin further increased LBWR and density of Ki-67-positive cells but also fetal mortality. TO with or without adjuncts induced elastin deposits at the tips of arising secondary crests, increased air space size, and decreased septal thickness. CONCLUSION TO had effects on lung proliferation and advanced the morphologic appearance. Addition of albumin increased density of proliferating cells and LBWR, yet at the expense of additional fetal loss.

OP02.01: Long term pulmonary gene therapy with a lentiviral vector in a fetal rat model

DNA Blood Mini kit was used to isolate extracellular DNA from maternal plasma samples. Results: Mean values of tcf DNA in the GLO assay in Group A were 6327.3 genome equivalents (GE)/mL, in Group B 1734.9 GE/mL and in Group C 910.6 GE/mL. Mean values of the tcf DNA in the GADPH assay in Group A were 7332.1 GE/mL, in Group B 2357.2 GE/mL and in Group C 1280.1 GE/mL. Mean values of the fcf DNA in the SRY assay in Group A were 326.9 GE/mL, in Group B 63.1 GE/mL and in Group C 31.6 GE/mL. Conclusions: The amounts of both tcf DNA and fcf DNA were significantly higher in pregnancies with placenta associated disorders compared to controls in all performed assays. We intend to perform further studies to assess whether quantification of extracellular DNA could be used as a non-invasive screening test for placenta associated disorders earlier in pregnancy.