Krabbe disease: Combined therapies for the central and peripheral nervous systems

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This is a proposal to continue our studies on the treatment of the mouse models of Krabbe disease or globoid cell leukodystrophy (GLD), an autosomal recessive disorder caused by the deficiency of galactocerebrosidase (GALC) activity. GALC is responsible for the lysosomal catabolism of galactosylceramide and psychosine. In GLD, the accumulation of psychosine in the CNS and PNS results in the apoptotic death of myelin-forming cells. Other mechanisms, including activation of resident microglial cells, recruitment of blood macrophages and inflammation, also play important roles in the pathogenesis. The only treatment currently available for some patients is hematopoietic stem cell transplantation (HSCT). While this treatment helps to maintain cognitive abilities, motor function remains a problem. Therapy trials with viral vectors and HSCT in the dog and mouse models have not lead to complete clinical, biochemical and pathological correction. It is clear that combined therapy will be required for entirely successful treatment. Questions related to early pathological changes and timing and dosage of GALC activity remain to be answered. In Aim 1, we will generate transgenic mice that have GALC expression under control of a tetracycline (tet) responsive promoter. Initially, we will produce Tet-Off mice (trG) that will have high GALC expression in the absence of tet analogues. After detailed characterization of these mice, they will be mated with heterozygous GALC-deficient mice to produce mice with no GALC activity except that regulated by tet analogues. The trG mice will be used to answer important questions regarding the timing of treatment and dosage of GALC activity needed for correction, and as a source of stem cells for transplantation. In Aim 2, we will improve upon the outcome of BMT by preparing and testing viral vectors for in vivo and ex vivo therapy trials. We have prepared a high titer lentiviral vector both for raising GALC activity in bone marrow cells for transplantation and for direct intracerebral injection. In addition we will construct a lentiviral vector containing both the mouse GALC gene and a tet responsive element. This vector will be used to transduce mouse BM cells that will have GALC high activity, above the low levels present in untransduced cells from normal donor mice and, in addition, will have the expression of GALC under control of tet analogues. We will determine the ability of GALC-AAV2/1 injected into the hind limb muscles to attain high GALC expression in order to correct the residual motor deficits in mice receiving BMT. Also, intracerebral, intraventricular and intracerebellar injections of mGALC-AAV2/1 will be combined with BMT and approved drugs that could reduce inflammation and prevent apoptosis. Initially these drugs will include minocycline and Indomethacin. These drugs have been successful in reducing demyelination and in providing neuroprotection in other neurodegenerative disorders. Studies using the dog model of GLD have been dropped from this application. Krabbe disease a genetic disease affecting mostly infants. Treatment options are limited at this time, but the studies proposed in animal models could lead the way to improved therapies for human patients. A combination of therapies may be needed to both stop the pathological events and provide long-term correction.

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