Fetal enzyme replacement and stem cell transplantation in murine Sly syndrome targeting microglia

Mucopolysaccharidosis type 7 (MPS7) is a lysosomal storage disorder resulting in multi-organ dysfunction and in utero demise. Post-natal enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are unable to address neurologic manifestations because of the blood-brain barrier (BBB). We study whether in utero ERT (IUERT) or HSCT (IUHSCT) can penetrate the BBB, targeting microglia as they are enriched in the missing enzyme. Prior IUERT work showed neurologic benefit - we now aim to analyze the extent of microglial engraftment, and to provide a permanent enzyme source with IUHSCT. We treated MPS7 fetuses at E14.5 with IUERT or IUHSCT. Following injection of missing glucuronidase (GUS), performed flow cytometry for enzyme activity on brain at E18. Controls included GUS injection in adult mice. We stained brains for CD68 (to quantify microglial inflammation). We bred CX3CR1-GFP donor mice (engrafted microglia express CX3CR1 and are therefore green on histology) and transplanted fetal liver mononuclear cells, initially into wild-type mice. Chimeric brains then underwent immunofluorescent staining for microglial markers. We injected 46 fetuses with enzyme or PBS control. 6 of 7 treated homozygous fetuses had detectable enzyme activity (24.6 ± 9.5% of microglia, some with wild-type activity levels). Accordingly, CD68 staining showed decreased brain inflammation. In contrast, 6 adult homozygous mice were treated, and none developed detectable brain GUS activity. We transplanted 5 litters of wild-type mice, and 9 mice engrafted (8.5±2.2% multilineage chimerism in blood and bone marrow). In chimeric mice at 3 weeks, transplanted stem cells co-stained with GFP and Iba1, indicating engraftment into the brain as microglia. These experiments suggest that IUERT can penetrate the BBB and decrease brain inflammation. IUHSCT results in microglial engraftment and increasing chimerism may further improve results. These findings support in utero therapy for MPS7, for which we are currently developing a clinical protocol.