Identification of modifications to the maternal-fetal interface transcriptome associated with placental insufficiency and a novel gene therapy for the treatment of fetal growth restriction (Placenta)

Fetal growth restriction (FGR) affects between 5-10% of all live births. Placental insufficiency is a leading cause of FGR, resulting in reduced nutrient and oxygen delivery to the fetus. Currently, there is no effective in utero treatment options for FGR, or placental insufficiency. We have developed a gene therapy to deliver human insulin-like 1 growth factor (hIGF1) to the placenta via a non-viral nanoparticle delivery mechanism as potential treatment of FGR. Using a guinea pig maternal nutrient restriction (MNR) model of FGR, we aimed to understand the transcriptional changes within the placenta associated with placental insufficiency that occur at the beginning stages of FGR (mid-pregnancy), and the immediate impact of hIGF1 nanoparticle treatment on the placental transcriptome. Using RNAsequencing, we analyzed protein coding genes of three experimental groups: Control dams and MNR receiving a sham treatment, and MNR dams receiving hIGF1 nanoparticle treatment. Pathway enrichment analysis comparing MNR placentas to Control revealed upregulation of pathways associated with degradation and repair of genetic information and downregulation of pathways associated with transmembrane transport. Similarly, differentially expressed genes that were decreased in MNR + hIGF1 placentas compared to Control demonstrated downregulation in pathways relating to transporter activities, but upregulation in pathways associated with positive regulation of phosphorylation and kinase activity. When compared to MNR placentas, MNR + hIGF1 placentas demonstrated changes to genes associated with transmembrane transporter activity including ion, vitamin and solute carrier (SLC-mediated) transport. Overall, this study identifies the key signaling and metabolic changes occurring in the placenta that contribute to placental insufficiency, and increases our understanding of the pathways that increasing placental IGF1 expression acts on and corrects. Overall design: Female (dams) Dunkin-Hartley guinea pigs were assigned to either the control diet group (n = 4) or MNR diet group (n = 7). Control dams were provided food and water was ad libitum; MNR dams were provided water ad libitum however, food intake was restricted to 70% per kilogram body weight of the Control group. At GD30-33, dams underwent an ultrasound-guided, transuterine, intra-placental injection of either sham (Control and MNR) or hIGF1 nanoparticle (MNR). Dams were sacrificed five (GD35-38) days after hIGF1 nanoparticle treatment. Placenta tissue from two fetuses per litter (one female and one male) was blunt dissected away from the rest of the maternal-fetal interface and snap-frozen in liquid nitrogen for RNA extraction. RNA-Seq Libraries for each gender and experimental group were generated using the Illumina Stranded mRNA Prep Kit. RNA-Seq Libraries were sequenced using the Illumina NovaSeq platform.

胎儿生长受限（Fetal growth restriction, FGR）影响约5%-10%的活产儿。胎盘功能不全是FGR的主要诱因，会导致胎儿获取的营养与氧气供应减少。目前尚无针对FGR或胎盘功能不全的有效宫内治疗手段。本团队开发了一种基因疗法，通过非病毒纳米颗粒递送系统将人胰岛素样生长因子1（human insulin-like 1 growth factor, hIGF1）递送至胎盘，作为FGR的潜在治疗方案。本研究利用豚鼠母体营养限制（maternal nutrient restriction, MNR）诱导的FGR模型，旨在探究FGR早期（妊娠中期）胎盘功能不全相关的胎盘转录组变化，以及hIGF1纳米颗粒治疗对胎盘转录组的即时影响。通过RNA测序（RNA sequencing, RNA-seq），我们分析了三个实验组的蛋白编码基因：正常饮食对照组孕鼠、接受假处理的MNR组孕鼠，以及接受hIGF1纳米颗粒治疗的MNR组孕鼠。将MNR组胎盘与对照组胎盘进行通路富集分析，结果显示与遗传信息降解及修复相关的通路出现上调，而与跨膜转运相关的通路则呈下调趋势。同样，与对照组相比，MNR+hIGF1组胎盘中表达下调的差异基因，其富集通路与转运活性相关，但与磷酸化正调控及激酶活性相关的通路则出现上调。将MNR+hIGF1组胎盘与MNR组胎盘相较，可见与跨膜转运活性相关的基因发生显著改变，涵盖离子、维生素及溶质载体（SLC介导）转运相关基因。综上，本研究明确了胎盘功能不全发生时胎盘中关键的信号转导与代谢变化，并加深了我们对胎盘IGF1表达上调所作用并予以纠正的通路的认知。整体实验设计：将雌性邓金-哈特利（Dunkin-Hartley）豚鼠孕鼠分为正常饮食对照组（n=4）与MNR饮食组（n=7）。对照组孕鼠可自由进食饮水；MNR组孕鼠可自由饮水，但进食量被限制为对照组每千克体重摄食量的70%。于妊娠第30-33天（GD30-33），通过超声引导经子宫行胎盘内注射，为对照组与MNR组孕鼠给予假处理，为MNR组孕鼠给予hIGF1纳米颗粒。在hIGF1纳米颗粒治疗5天后（妊娠第35-38天，GD35-38），处死孕鼠。每窝选取2只胎鼠（1雌1雄）的胎盘组织，从母胎界面钝性分离后，置于液氮中快速冷冻以用于RNA提取。使用Illumina Stranded mRNA Prep Kit构建各性别及实验组的RNA测序文库，并通过Illumina NovaSeq平台完成测序。