Targeted conditioning to maximize prenatal HSC engraftment for SCD

项目摘要： 通过宫内移植(IUTx)治疗遗传性疾病已经安全地进行了几十年 人类。在美国，第一例IUTx治疗使用了造血干细胞(HSC)，并纠正了一名患有X-SCID的儿童。 从这一开创性的时刻开始&gt；25年。以前，50名患者现在已经接受了14次这种手术 不同的遗传病。然而，由于仍不清楚的原因，完全的治疗成功已经 仅在X-SCID患者中实现。因此，更好地理解HSC 在需要IUTx后，植入会受到阻碍，因此可以制定策略来达到治疗水平 在可能受益于IUTx的其他遗传性疾病中，如血红蛋白疾病中，HSC植入的可能性很大。我们 其他人发现了发育中的胎儿的几个特征，这些特征可能会对其能力产生负面影响 作为顺从的HSC接受者。这些因素包括来自高增殖宿主HSC的竞争， 比最初所知的更显著的胎儿免疫障碍，以及新生儿的成熟度和接受度 供体(成人)造血干细胞植入所需的骨髓壁龛。在这里，我们以胎羊为模型，建议： (目标1)定义植入障碍的性质，并通过使用非遗传毒性调节来克服植入障碍 剖析生态位可获得性、宿主HSC竞争和胎儿免疫在成人移植中的作用 IUTx后供者的HSC，以及(目标2)确定供者的表型和功能的影响 HSC对IUTx植入水平的影响。我们希望，在完成前两个目标后，我们将 不仅已经确定了抵抗造血干细胞移植的机制，而且我们还将 达到了20%-25%的HSC植入的最低目标，这将使IUTX成为一种可行的治疗方法 治疗血红蛋白疾病的方法。其中，镰状细胞病(SCD)是最常见的遗传性血液 在美国，这是一种可以从IUTx中受益的疾病，因为即使胎儿受到保护 从胎儿血红蛋白(Hb)出现的病状开始，SCD的临床表现始于婴儿早期， 使儿童面临中风、脾危机、疼痛发作、危及生命的感染等并发症的风险， 以及急性胸部综合征的发作，这可能会导致永久性的肺损伤。与SCD有直接关联， 绵羊表现出与人类相同的胎儿到成人Hb转换的发育模式。最近，使用CRISPR/ CAS编辑和随后的体细胞核移植，我们产生了一种具有疾病表型的SCD绵羊 与人类患者相似，在血涂片中显示镰状细胞，Hb溶解试验阳性，HBs Hb电泳法检测。在目标3中，我们建议通过监测绵羊的SCD模型来验证 动物随着时间的推移，确定导致镰状细胞危机的应激源，并定义急性和慢性疾病 并发症。此外，我们还将测试IUTx治疗/治疗SCD的疗效。完成后， 我们希望这些研究将有助于开发新的策略，以达到HSC的治愈水平 在IUTX后植入，并将验证一个对SCD社区的高度临床相关性的模型。

PROJECT ABSTRACT: Treatment of genetic disorders by in utero transplantation (IUTx) has safely been performed for decades in humans. The first IUTx cure, in the US, used hematopoietic stem cells (HSC) and corrected a child with X-SCID. Since this groundbreaking moment >25 yrs. ago, >50 patients have now been treated with this procedure for 14 different genetic disorders. However, for reasons that are still not well understood, full therapeutic success has only been achieved in X-SCID patients. Thus, a better understanding of the mechanisms by which HSC engraftment is hindered after IUTx is required, so that strategies can be developed to achieve therapeutic levels of HSC engraftment in other genetic disorders, such as hemoglobinopathies, that could benefit from IUTx. We and others have identified several characteristics of the developing fetus that may negatively impact its ability to serve as an amenable HSC recipient. Among these factors are competition from highly proliferative host HSC, more significant fetal immune barriers than initially known, and the degree of maturity and receptivity of nascent BM niches required for engraftment of donor (adult) HSC. Here, using fetal sheep as a model, we propose to: (Aim1) define the nature of, and overcome, the barriers to engraftment by using non-genotoxic conditioning to dissect the role that niche availability, host HSC competition, and fetal immunity play in the engraftment of adult donor HSC following IUTx, and (Aim 2) determine the impact of the phenotype and functionality of the donor HSC on the levels of engraftment following IUTx. We hope that, upon completion of these first 2 Aims, we will not only have identified the mechanisms involved in resistance to HSC engraftment, but we will also have achieved a minimum target of 20-25% HSC engraftment, which would allow IUTX to become a viable therapeutic approach for hemoglobinopathies. Among these, sickle cell disease (SCD) is the most common inherited blood disorder in the US, and one of the diseases that could benefit from IUTx, since even though the fetus is protected from sickling by the presence of fetal hemoglobin (Hb), clinical manifestations of SCD start during early infancy, placing the child at risk of complications such as stroke, splenic crisis, pain episodes, life-threatening infections, and episodes of acute chest syndrome, which can cause permanent lung damage. Of direct relevance to SCD, sheep exhibit the same developmental pattern of fetal to adult Hb switching as humans. Recently, using CRISPR/ Cas editing and subsequent somatic cell nuclear transfer, we produced SCD sheep with a disease phenotype mirroring that of human patients, displaying sickled cells in blood smears, positive Hb solubility test, and HbS detected by Hb electrophoresis. In Aim 3, we propose to validate the sheep SCD model by monitoring the animals over time, determining the stressors that induce sickle cell crises, and defining acute and chronic disease complications. In addition, we will test the therapeutic efficacy of IUTx for treating/curing SCD. Upon completion, we hope these studies will contribute to the development of novel strategies to achieve curative levels of HSC engraftment after IUTX and will validate a highly clinically relevant model for the SCD community in general.