Prenatal Cell and Gene Therapy for Hemophilia A

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Project Summary Intrauterine stem cell transplantation (IUTx) using a minimally invasive, ultrasound-guided approach has been performed in ~50 human patients for 14 different genetic disorders, proving that this procedure poses minimal risk to both the fetus and the mother. Hemophilia A (HA) is an ideal disease to be treated by IUTx, since 75% of HA cases can be diagnosed through prenatal screening. IUTx could be curative, or at least convert severe HA to a moderate/mild phenotype, significantly reducing the need for FVIII infusions. Importantly, exposure to vector-encoded FVIII during early immunologic development would induce tolerance FVIII and avoid formation of inhibitors, the most serious and threatening complication in HA treatment. Indeed, recent studies showed in both severe and non-severe HA patients, that the development of FVIII inhibitors is associated with increased risk of death. Using the same preclinical animal model, that allowed the delineation of the conditions used in clinical IUTx, we showed that prenatal infusion of transduced bone marrow stromal progenitor cells (MSPs) resulted in robust long-term, multi-organ integration into both parenchyma and vasculature, long-term release of secreted transgene products into the circulation, and development of immune tolerance. Using the same sheep model, similar results were obtained after IUTx of amniotic fluid-derived stromal progenitors (AFPs). Utilizing a line of sheep that emulates the genetics, inhibitor formation, and clinical symptoms of the severe form of human HA, we showed that the postnatal transplantation of paternal (haploidentical) MSPs engineered to express high levels of FVIII led to complete phenotypic correction of two pediatric HA sheep, reversal of existing hemarthroses, and return to normal physical activity. However, like with FVIII infusions in human patients, these two sheep developed FVIII inhibitors following postnatal treatment. This work thus provides compelling evidence that the transplantation of FVIII-expressing stromal cells could be curative for HA, if inhibitor formation could be avoided. Therefore, we hypothesize that using an IUTx-based strategy to treat HA will overcome the two major shortcomings that have been observed following factor replacement therapy and current gene therapy approaches to treat HA, namely a lack of sustained FVIII expression and immunologic responses to the therapeutic protein. We propose to: 1) determine the ideal cell source to obtain the highest long-term engraftment levels and the in vivo distribution patterns that maximize release of FVIII into the circulation; 2) use HA sheep to demonstrate that an IUTx approach using the optimal cell source and FVIII transgene construct, can be curative, or at least permanently convert severe HA to a mild phenotype; 3) test whether receiving IUTx precludes inhibitor induction following postnatal FVIII infusion and/or induces tolerance that persists even after postnatal challenge with FVIII in adjuvant. Upon completion, these studies will prove the safety and efficacy of using cells as a FVIII delivery platform, and demonstrate the ability of IUTx to cure or improve HA phenotype, and defeat the immune-related hurdles that currently hinder clinical HA treatment.