Targeted conditioning to maximize prenatal HSC engraftment for SCD

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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.