Severe fetal growth restriction (FGR) affects 1:500 pregnancies, is untreatable and causes serious neonatal morbidity and death. Reduced uterine blood flow (UBF) and lack of bioavailable VEGF due to placental insufficiency is a major cause. Transduction of uterine arteries in normal or FGR sheep and guinea pigs using an adenovirus (Ad) encoding VEGF isoforms A (Ad.VEGF-A₁₆₅) and a FLAG-tagged pre-processed short form D (D^ΔNΔC, Ad.VEGF-D^ΔNΔC-FLAG) increases endothelial nitric oxide expression, enhances relaxation and reduces constriction of the uterine arteries and their branches. UBF and angiogenesis are increased long term, improving fetal growth in utero. For clinical trial development we compared Ad.VEGF vector transduction efficiency and function in endothelial cells (ECs) derived from different species. We aimed to compare the transduction efficiency and function of the pre-clinical study Ad. constructs (Ad.VEGF-A₁₆₅, Ad.VEGF-D^ΔNΔC-FLAG) with the intended clinical trial construct (Ad.VEGF-D^ΔNΔC) where the FLAG tag is removed. We infected ECs from human umbilical vein, pregnant sheep uterine artery, pregnant guinea pig aorta and non-pregnant rabbit aorta, with increasing multiplicity of infection (MOI) for 24 or 48 hours of three Ad.VEGF vectors, compared to control Ad. containing the LacZ gene (Ad.LacZ). VEGF supernatant expression was analysed by ELISA. Functional assessment used tube formation assay and Erk-Akt phosphorylation by ELISA. VEGF expression was higher after Ad.VEGF-D^ΔNΔC-FLAG and Ad.VEGF-D^ΔNΔC transduction compared to Ad.VEGF-A₁₆₅ in all EC types (*p < 0.001). Tube formation was higher in ECs transduced with Ad.VEGF-D^ΔNΔC in all species compared to other constructs (***p < 0.001, *p < 0.05 with rabbit aortic ECs). Phospho-Erk and phospho-Akt assays displayed no differences between the three vector constructs, whose effect was, as in other experiments, higher than Ad.LacZ (***p < 0.001). In conclusion, we observed high transduction efficiency and functional effects of Ad.VEGF-D^ΔNΔC vector with comparability in major pathway activation to constructs used in pre-clinical studies, supporting its use in a clinical trial.

中文翻译：

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血管内皮生长因子腺病毒载体在内皮细胞中用于胎盘功能不全基因治疗的效率和功能比较


严重胎儿生长受限 (FGR) 影响 1:500 的妊娠，无法治疗，会导致严重的新生儿发病和死亡。主要原因是胎盘功能不全导致子宫血流量 (UBF) 减少和生物可利用的 VEGF 缺乏。使用编码VEGF同工型 A (Ad.VEGF-A ₁₆₅ ) 的腺病毒 (Ad) 和带 FLAG 标记的预加工短型 D (D ^ΔNΔC 、 Ad.VEGF-D) 转导正常或 FGR 绵羊和豚鼠的子宫动脉^ΔNΔC -FLAG) 增加内皮一氧化氮表达，增强松弛并减少子宫动脉及其分支的收缩。 UBF 和血管生成长期增加，改善胎儿在子宫内的生长。为了进行临床试验开发，我们比较了 Ad.VEGF 载体在不同物种的内皮细胞 (EC) 中的转导效率和功能。我们的目的是比较临床前研究Ad的转导效率和功能。构建体（Ad.VEGF-A ₁₆₅ 、Ad.VEGF-D ^ΔNΔC -FLAG）与预期的临床试验构建体（Ad.VEGF-D ^ΔNΔC ）（其中FLAG标签被去除）。我们感染了来自人脐静脉、怀孕绵羊子宫动脉、怀孕豚鼠主动脉和非怀孕兔主动脉的 EC，与对照 Ad 相比，三种 Ad.VEGF 载体在 24 或 48 小时内感染复数 (MOI) 不断增加。含有LacZ基因 (Ad.LacZ)。通过ELISA分析VEGF上清液表达。功能评估使用管形成测定和 ELISA 的 Erk-Akt 磷酸化。与 Ad 相比，Ad.VEGF-D ^ΔNΔC -FLAG 和 Ad.VEGF-D ^ΔNΔC转导后 VEGF 表达较高。所有 EC 类型中 VEGF-A_{均为 165} (* p < 0.001)。与其他构建体相比，所有物种中用 Ad.VEGF-D ^ΔNΔC转导的 EC 的管形成率更高（*** p < 0.001，* p < 0.05，兔主动脉 EC）。 Phospho-Erk 和磷酸化 Akt 测定显示三种载体构建体之间没有差异，与其他实验一样，其效果高于 Ad.LacZ (*** p < 0.001)。总之，我们观察到 Ad.VEGF-D ^ΔNΔC载体的高转导效率和功能效果，并且在主要途径激活方面与临床前研究中使用的构建体具有可比性，支持其在临床试验中的使用。