Abstract
Mucopolysaccharidosis Type VII (MPS VII) is one of many lysosomal storage diseases and is caused by a deficiency of beta-glucuronidase (GUS). Progressive accumulation of undegraded glycosaminoglycan (GAG) intermediates occurs in the lysosome diminishing intellect, mobility, organ function, and life span. Previously, the efficacy of enzyme replacement therapy (ERT), bone marrow transplantation, and gene therapy have been tested in MPS VII mice. GUS supplied to serum by ERT or released into extracellular spaces from transplanted (Tx) normal or gene transduced cells is taken up by receptor mediated endocytosis and transported to the lysosome. All these treatments reduce lysosomal storage in visceral organs, but poorly deliver sufficient enzyme to the central nervous system (CNS). We hypothesized that in utero intrathecal transplantation of primary neuronal stem cells (NSC) would remedy the GUS deficiency of MPS VII CNS. We isolated NSC from the cortical ventricular zone (CVZ) of E14 GUS+ eGFP+ fetuses. CVZ NSC incubated with EGF and bFGF formed neurospheres in 3–5 days. E14 MPS VII GUS- recipients were Tx intrathecally with NSC and examined for engraftment by histological staining for GUS and eGFP expressing cells. Successful engraftment occurred in 13 of 23 (56.5%) MPS VII recipients. Three animals Tx with 5–25,000 (K) had GUS+ cells in the rostral migratory stream (RMS). Two of seven animals Tx with 50-80K had donor cells in the RMS and five had donor cells in olfactory bulb, cerebrum (cortical layers, corpus callosum and striatum), midbrain, hippocampus (including dentate gyrus), and the cerebellum (Purkinje layers, peduncle, and lobular regions). This demonstrates “complete” dispersal. One mouse Tx with 90K had donor cells lining the lateral ventricle. Another Tx with 160K had donor cells lining ventricular spaces and throughout the cerebrum away from the RMS. An animal Tx with 180K NSC had “complete” donor engraftment like those above. Histological staining for GUS+ donor cells in mice with “complete” engraftment showed levels of staining greater than untreated heterozygous controls, both in numbers of cells positive and in intensity of staining. Biochemistry of tissue sections from these animals averaged 59.5 ±9.2% (mean ±SE) of normal (+/+) activity. The oldest animal detected with “complete” engraftment thus far was 7 months old at sacrifice, demonstrating long-term durability of engraftment. Using anti-eGFP, beta-tubulin III (neurons), and GFAP (astrocytes) antibodies, we confirmed the “complete” engraftment was due to widespread dispersal and integration of donor derived cells and not to diffusion of GUS enzyme from a few engrafted cells. The lineage markers confirmed normal differentiation of donor CVZ NSC. Further evaluations are underway to measure cognitive function in treated animals by the Repeated Acquisition and Performance Chamber (RAPC). Comparison of untreated MPS VII adult mice to normal controls by the RAPC indicates significant deficiencies in learning and memory in untreated MPS VII mice and confirms our ability to measure functional changes in treated animals. In summary, GUS+ eGFP+ fetal NSC primary isolates engraft brain of MPS VII fetal recipients and restore GUS activity ≥ phenotypically normal MPS VII heterozygotes.
Hearing Loss in Mucopolysaccharidoses: Current Knowledge and Future Directions