We are investigating cell-based gene transfer approaches to treat the central nervous system (CNS) component of lysosomal storage disorders. Animal models of mucopolysaccharidosis (MPS) VII (ly disease), beta- glucuronidase (GUSB) deficiency, are used as test systems. Treatment is based on lysosomal enzymes being secreted from genetically corrected cells, diffusing through tissue, and being taken up by mutant cells to restore the missing enzymatic activity. We have demonstrated that gene therapy can work, in principle, in the brains of MPS VII mice. However, there are still substantial barriers to achieving permanent and complete correction, particularly of the global lesions that occur in the brain. We have made significant progress towards solving some of the major problems by: 1) developing a retrovirus vector, a noel cell isolation method, which each appear to overcome the problem of vector extinction in vivo; 2) demonstrating that vector-expressed normal enzyme can diffuse through neural tissue and correct affected neurons at a distance; and 3) showing that a neural stem cell line can deliver enzyme widely in the host brain and differentiate into normal brain elements, and developing new neural stem cell lines to test gene delivery strategies. For the next grant period, we propose to use the MPS VII mouse brain to: 1) measure the sphere of enzyme diffusion that can be achieved from grafts that are continuously secreting high levels, and determine the extent of phenotypic correction, 2) determine if delivery to the diseased brain cells can be improved by engineering the enzyme protein; and 3) determine if syngeneic neural stem cells, transduced with our new vector, can distribute therapeutic enzyme in a global fashion. We will also test the methods in MPS VII dogs to measure the distribution of enzyme in a brain which is 200 times larger in volume than a mouse brain. These studies will define the limitations that are likely to be encountered in trying to treat an affected child's brain, which is only about 1o times larger than the dog brain.
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