Targeted conditioning to maximize prenatal HSC engraftment for SCD

项目摘要： 通过子宫内移植（IUTx）治疗遗传性疾病已安全进行了数十年， 人类在美国，第一个IUTx治疗使用造血干细胞（HSC）并纠正了患有X-SCID的儿童。 自25年前这一突破性时刻以来，已有超过50名患者接受了14年的治疗。 不同的遗传疾病然而，由于尚不清楚的原因，完全的治疗成功 仅在X-SCID患者中实现。因此，更好地了解HSC 在需要IUTx后，植入受阻，因此可以制定策略以达到治疗水平 HSC植入其他遗传性疾病，如血红蛋白病，可以受益于IUTx。我们 和其他人已经确定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， 作为一个合格的HSC接受者。这些因素包括来自高度增殖的宿主HSC的竞争， 比最初已知的更显著的胎儿免疫屏障，以及新生儿的成熟程度和接受性， 供体（成人）HSC植入所需的BM龛。在这里，使用胎羊作为模型，我们建议： （目标1）通过使用非遗传毒性条件处理来定义植入障碍的性质并克服植入障碍， 剖析生态位可用性，宿主HSC竞争和胎儿免疫在成体移植中的作用。 IUTx后供体HSC，以及（目的2）确定供体表型和功能的影响 HSC对IUTx后植入水平的影响。我们希望，在完成前两个目标后， 我们不仅已经确定了HSC移植抵抗的机制，而且我们还将 实现了20 - 25% HSC植入的最低目标，这将使IUTX成为一种可行的治疗方法， 血红蛋白病的方法。其中，镰状细胞病（SCD）是最常见的遗传性血液病。 这种疾病在美国，也是可以从IUTx中受益的疾病之一，因为即使胎儿受到保护， 由于胎儿血红蛋白（Hb）的存在而形成镰状，SCD的临床表现在婴儿早期开始， 使儿童面临并发症的风险，如中风、脾危象、疼痛发作、危及生命的感染， 和急性胸部综合征的发作，这可能导致永久性肺损伤。与SCD直接相关， 绵羊表现出与人类相同的从胎儿到成人Hb转换的发育模式。最近，利用CRISPR/ Cas编辑和随后的体细胞核移植，我们产生了具有疾病表型的SCD绵羊。 反映了人类患者的情况，在血涂片中显示镰状细胞，Hb溶解度试验阳性，HbS 通过Hb电泳检测。在目标3中，我们建议通过监测 动物随着时间的推移，确定诱导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，我们将测试IUTx治疗/治愈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，以达到治疗HSC的水平 IUTX后的移植，并将验证一个高度临床相关的模型，为SCD社区一般。

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.