INSTINCT Study: Intranasal Stem Cells for Improving Neurodevelopmental Outcomes in Neonatal Encephalopathy

出生时的问题导致婴儿缺氧会导致称为新生儿脑病（NE）的脑功能紊乱，这可能导致长期脑损伤和脑瘫。在出生后尽快将婴儿冷却3摄氏度三天已被证明是安全的，并导致长期的大脑保护和改善的结果，甚至到学龄。冷却得到NICE的认可，并在英国的新生儿重症监护病房中用于中度至重度NE。然而，尽管冷却是NE婴儿的一个重要进步，但44-53%的婴儿仍然有不良后果。这可能是因为冷却可能对严重损伤或先前暴露于感染和炎症的效果较差。非常需要补充干预措施来改善冷却和不冷却的结果。在过去的10年里，已经研究了几种治疗药物作为婴儿HT的预防剂，例如促红细胞生成素、褪黑激素、别嘌呤醇，但是目前除了用于NE的冷却之外没有治疗方法。使用干细胞成功治疗新生儿脑损伤正在成为一种有前途的治疗方法。人脐带间充质干细胞（human umbilical cord mesenchymal stem cells，huMSC）具有自我更新和刺激宿主脑细胞再生和修复的功能，具有比成人组织来源的MSC更上级的抗炎特性。重要的是，尽管huMSC不能长期存活并自身替换受损组织，但它们通过分泌生长因子、细胞因子和细胞外囊泡（EV）来调节损伤和修复，从而对缺血性脑环境的需要做出反应。huMSC的这些内在适应性特性使它们成为治疗NE的破坏性作用的优秀候选者。新生儿大脑仍处于发育活跃期，导致huMSC的高效率。在NE的试点临床前模型中，我们观察到在缺氧一段时间后24小时鼻内给予2剂3000万huMSC的脑保护作用，而静脉内huMSC没有保护作用。我们能够在脑组织中检测到这些huMSC。来自男性受试者的这些数据表明，huMSC（鼻内给予）基于临床相关标志物（aEEG和脑能量代谢的恢复）增强脑保护。对于临床转化，关于huMSC在常温和低温环境中是否具有保护作用以及男性和女性是否存在不同的神经保护反应的数据不足。最近，EV已被确定为干细胞旁分泌信号传导的关键介质。EV以蛋白质、脂质和核酸的形式递送其内容物;它们克服了细胞疗法的一些限制，包括更容易储存、低免疫原性、通过小血管和无致瘤潜力。我们提出了以下3个里程碑，为将来的NE婴儿临床试验做准备。第一个里程碑将准备huMSC和EV。我们可以使用UCL生产的临床级huMSC。第二个也是关键的里程碑将评估男性和女性受试者在缺氧后12小时和36小时鼻内给予2剂huMSC的安全性和疗效，有和没有冷却。我们将根据aEEG恢复、脑能量代谢和组织学评估结局。如果是积极的，我们将满足MHRA的目的是转移到临床试验。第三个里程碑将评估缺氧后12和36小时鼻内给予2剂EV的安全性和有效性。将使用产生相当于3000万huMSC的抗炎作用的EV体积（先前在里程碑2b中的体外研究中确定）。这将为MSC治疗的有益作用介导的机制提供信息，并可能导致改善未来NE婴儿临床试验的策略。

Problems around the time of birth causing a lack of oxygen to the baby can cause disordered brain function called neonatal encephalopathy (NE), which can result in long term brain damage and cerebral palsy. Cooling a baby by 3 degrees Celsius for three days as soon as possible after birth has been shown to be safe and leads to long term brain protection and improved outcome even to school age. Cooling is endorsed by NICE and adopted in neonatal intensive care units in UK for moderate to severe NE. However, although cooling is a major step forward for babies with NE, 44-53% still have adverse outcomes. This may be because cooling may be less effective with severe injury or there was prior exposure to infection and inflammation. Supplemental interventions to improve outcomes with and without cooling are greatly needed. Over the last 10 years, several therapeutic agents have been studied as adjuncts to HT for babies, such as erythropoietin, melatonin, allopurinol, but there is currently no therapy apart from cooling for NE. The use of stem cells to successfully treat neonatal brain injury is emerging as a promising therapy. Human umbilical cord mesenchymal stem cells (huMSC) self renew and stimulate host brain cells to regenerate and repair, with superior anti-inflammatory properties than MSC from adult tissues. Importantly, although huMSC do not survive long term and replace damaged tissues themselves, they react to the needs of the ischemic cerebral environment by secretion of growth factors, cytokines and extracellular vesicles (EVs) to regulate damage and repair. These intrinsic adaptive properties of huMSC make them excellent candidates to treat the devastating effects of NE. The newborn brain is still in a developmentally active phase, leading to high efficiency of huMSC.In a pilot pre-clinical model of NE, we observed brain protection with 2 doses of 30 million huMSC given intranasally 24h after a period of oxygen deprivation, whereas intravenous huMSC were not protective. We were able to detect these huMSC within the brain tissue. These data from male subjects suggest that huMSC (given intranasally) augment brain protection based on clinically relevant markers (recovery of aEEG and brain energy metabolism). For clinical translation, there is insufficient data on whether huMSC are protective in both normothermic and hypothermic milieu and whether there is different neuroprotective response from males and females. Recently, EVs have been identified as the key mediators of stem cell paracrine signalling. EVs deliver their contents in the form of proteins, lipids, and nucleic acids; they overcome some limitations of cell therapies, including easier storage, low immunogenicity, passage through small vessels and no tumorigenic potential. We propose the following 3 milestones as preparation for future clinical trials in babies with NE.The first milestone will prepare the huMSC and EVs. We have access to clinical-grade huMSC manufactured by UCL. The second and key milestone will assess safety and efficacy of 2 doses huMSC given intranasally at 12 and 36h after oxygen deprivation in both male and female subjects with and without cooling. We will assess outcome based on aEEG recovery, brain energy metabolism and histology. If positive we will meet MHRA with the aim to move to clinical trials. The third milestone will assess safety and efficacy of 2 doses of EVs given intranasally at 12 and 36h after oxygen deprivation. The volume of EVs that produce an equivalent anti-inflammatory effect as 30 million huMSC will be used (previously determined in vitro studies in milestone 2b). This will inform the mechanism(s) through which beneficial effects of MSC therapy are mediated and may lead to refining the strategy for future clinical trials in babies with NE.