Transcriptomics and biomarkers of fetal neuroinflammation

摘要 这是对HD98389(2019-24)的家长拨款的行政补充建议，标题为“消炎” 减少不良妊娠结局的药物目标“。父母资助的目标是发展Anakinra 重组人白介素1受体拮抗剂作为安全有效的宫内治疗药物 感染/炎症介导了早产和胎儿炎症。在父母的赠与中，怀孕的恒河猴 在一种宫内炎症(IUI)模型中，猕猴将被给予Anakinra。我们现在将利用 母基金的资源，以扩大母基金的目标。胎儿神经炎症与 随着脑瘫(CP)和自闭症谱系障碍(ASD)的后期发展，有显著的医学和 对公共健康的影响。作为父母赠款活动的一部分，我们已经证明了对IL1的抑制 使用Anakinra信号显著降低羊膜腔注射大肠杆菌引起的胎儿炎症 LP。我们现在已经开发出从冰冻的胎儿大脑样本中提取完整细胞核的能力，并能够 细胞测序。与胞质rna序列相比，核rna序列具有不需要酶消化的优点。 在37oC，从而消除了人为的基因表达。此外，核RNA序列可以在存档中完成 冷冻的大脑样本，从而节省了宝贵的动物使用，并降低了成本。在本行政副刊中 格兰特，我们将检验这样的假设，即Anakinra抑制IL1信号将降低区域特异性 羊膜腔内注射脂多糖诱导的胎儿神经炎症。我们建议从2开始做单核mRNA序列。 大脑的区域-脑室周围白质和前额叶皮质-与 CP和ASD的发病机制。我们将比较三组中区域特异性单核mrna序列。 恒河猴胚胎约80%孕期：1)对照组--羊膜内注射生理盐水，2)IA脂多糖， 3)IA LPS+Anakinra。所有的曝光量都是16小时，这是我们之前证明的最佳时间点 建议进行的研究。由父母资助的动物资源已经可用。因此，单个细胞 转录学研究将在资助年度内得到极大的便利和可行。这个胎儿的大脑 转录学研究将揭示IUI引起的神经炎症的发病机制， 并确定CP和ASD的生物标记物集的线索。这项研究还将进一步帮助临床前的发展 Anakinra作为IUI的治疗候选药物。

Summary This is an administrative supplement proposal to the parent grant HD98389 (2019-24) titled “Anti-inflammatory drug target to reduce adverse pregnancy outcomes”. The goal of the parent grant is to develop Anakinra (recombinant human IL1receptor antagonist) as a safe and effective therapeutic for intrauterine infection/inflammation mediated preterm labor and fetal inflammation. In the parent grant, pregnant Rhesus macaques will be given Anakinra in a model of intrauterine inflammation (IUI). We will now leverage the resources of the parent grant to expand the Aims of the parent grant. Fetal neuro-inflammation is associated with later development of cerebral palsy (CP) and autism spectrum disorders (ASD) with significant medical and public health implications. As part of the parent grant activities, we have demonstrated that inhibition of IL1 signaling using Anakinra significantly decreased fetal inflammation induced by intraamniotic injection of E. coli LPS. We have now developed capability of extracting intact nuclei from fetal frozen brain sample and to do single cell sequencing. Nuclear as compared to cytosolic RNA seq has the advantage of not needing protease digestion at 37oC thereby eliminating artifactual gene expression. Furthermore, nuclear RNA seq can be done in archived frozen brain sample thereby sparing precious animal use and reducing costs. In this administrative supplement grant, we will test the hypothesis that inhibition of IL1 signaling by Anakinra will decrease region specific fetal neuro inflammation induced by intraamniotic LPS. We propose doing single nuclear mRNA seq from 2 regions of the brain – the peri-ventricular white matter, and the prefrontal cortex – regions implicated in the pathogenesis of CP and ASD. We will compare region specific single nuclear mRNA seq in three groups of Rhesus macaque fetuses at about 80% gestation: 1) Controls – intraamniotic injection (IA) of saline, 2) IA LPS, 3) IA LPS + Anakinra. All exposures are for 16h, a time point that we previously demonstrated to be optimum for the proposed studies. The animal resources funded by the parent grant are already available. Thus, single cell transcriptomic studies will be greatly facilitated and feasible within the year of funding. This fetal brain transcriptomic study will reveal mechanistic insights in the pathogenesis of neuroinflammation resulting from IUI, and identify leads for biomarker sets for CP and ASD. The study will also further assist in pre-clinical development of Anakinra as a therapeutic candidate for IUI.