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THE YOUNG INVESTIGATOR'S PRIZE LECTURE 2013

Presented at the Neonatal Society 2013 Summer Meeting (programme).

David J Carr, University College London

Somatotrophic Effects of Prenatal Ad.VEGF Gene Therapy in the Growth-Restricted Sheep Fetus and Neonate

Background: Fetal growth restriction (FGR) is a leading cause of perinatal mortality and is associated with considerable morbidity in neonatal and later life. The commonest cause is reduced uterine blood flow (UBF). In normal sheep pregnancy, adenovirus (Ad) mediated over-expression of vascular endothelial growth factor (VEGF) in the uterine arteries (UtA) increases UBF. We hypothesised that enhancing UBF would improve fetal nutrient delivery in a sheep paradigm of FGR that is characterised by reduced UBF from mid-gestation.

Methods: Singleton pregnancies were established using embryo transfer in adolescent ewes subsequently overnourished to induce FGR (n=78) or control-fed (n=12). Ewes were randomised mid-gestation to receive bilateral UtA injections of 5x1011 particles Ad.VEGF-A165 or inactive treatment (5x1011 particles of control vector Ad.LacZ or saline). Fetal growth and wellbeing were evaluated using serial ultrasound by a single operator blind to study group. Late gestation study: UtA vasorelaxation and placental mRNA expression of various angiogenic factors/receptors were examined by organ bath analysis and qRT-PCR, respectively. Postnatal study: Pregnancies continued until spontaneous delivery near to term (=145 days). Lambs were weighed and measured at weekly intervals, and underwent immune and metabolic challenge, dual-energy X-ray absorptiometry (DEXA) and necropsy at 8, 9, 10 and 12 weeks of age, respectively. In both studies DNA methylation was quantified at 59 individual cytosine-guanine (CpG) dinucleotides in 14 CpG islands in ten somatotrophic axis genes [insulin, insulin-like growth factor (IGF)-1, IGF-2, H19, growth hormone (GH), glucocorticoid/insulin receptor (R), GHR, IGF1R and IGFR2] in hepatic tissues using bisulphite sequencing.

Results: In both studies, ultrasonographic fetal growth velocity was increased in Ad.VEGF-A165-treated vs. control-treated overnourished pregnancies at 3-4 weeks post-injection. At 0.9 gestation, fewer fetuses were markedly growth-restricted and fetal brain sparing was mitigated. Ad.VEGF-A165-transduced vessels showed enhanced vasorelaxation and FLT1/KDR expression was increased in the maternal placental compartment. Following delivery at 141±2.4 days (mean±SD, range 132–145 days) gestation Ad.VEGF-A165-treated lambs tended to be heavier (p=0.081) with increased placental efficiency (p=0.072). There were no differences in the level of neonatal care required to ensure lamb survival or acute phase response to immune challenge. Postnatal growth rates and glucose-stimulated insulin secretion were increased, however fractional growth and insulin resistance were unaffected. DEXA and post-mortem analysis demonstrated increased lean tissue mass. No epigenetic effects of Ad.VEGF-A165 were observed, but postnatally IGF-1 and insulin methylation were M>F and F>M, respectively, and insulin gene methylation correlated with fasting plasma insulin levels.

Conclusion: Ad.VEGF-A165 safely increases fetal growth in this ovine FGR paradigm and mitigates fetal brain sparing, an adaptation that is associated with poorer neurodevelopmental outcomes. Ad.VEGF-A165 increased UtA vascular reactivity and upregulated maternal placental FLT1/KDR expression, suggesting that its beneficial effects on fetal growth might be mediated at the placental level via effects on vascularity and/or nutrient transport capacity. Increased postnatal growth rates and insulin secretion in Ad.VEGF-A165-treated lambs most likely reflects their relative size advantage at birth rather than altered epigenetic status of key somatotrophic genes. We have recently secured funding to translate this potential therapy into the clinic. The EVERREST consortium will complete a full reproductive toxicology package and conduct a bioethical study and first in-woman I/IIa safety/efficacy trial of Ad.VEGF in pregnancies affected by severe early-onset FGR.

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