Lysosomal storage diseases (LSDs) comprise a large group of monogenic, metabolic disorders caused by deficiency of lysosomal hydrolases. Accumulation of intermediate metabolites in lysosomes affects systemic organs and the central nervous system (CNS). However, it is still largely unknown how an impaired lysosomal system influences cellular homeostasis and ultimately leads to organ dysfunction. The poor understanding of pathogenesis adds to the challenge in finding a treatment for LSDs that would be effective for both systemic and CNS disease. The long-term objective of the proposed research is to achieve a comprehensive evaluation of the CNS dysfunction that occurs in galactosialidosis (GS; protective protein/cathepsin A [PPCA deficiency] and GM1-gangliosidosis (GM1; beta-galactosidase [b-gal deficiency]). This knowledge will allow us to assess the feasibility, limitations, and effectiveness of specific therapies. To accomplish our goal, we have developed mouse models of cells that overexpress the correcting enzyme in a lineage specific manner effectively restore systemic organ function in GS mice, but only could partially ameliorate severely affected area of the CNS, such as the cerebellum. As a step toward developing more effective therapies for the CNS disease, we propose the following aims:
Aim 1. We will complete our ongoing studies of the phenotypes of GS and GM1 mice so that the pathogenesis of neurodegeneration can be elucidated. The combined use of histological staining and immunocytochemistry with antibodies to cellular and molecular markers for neurons and glia will enable us to determine the status and characteristics of cells at disease sites, and during disease progression.
Aim 2. We will generate transgenic mice in which expression of the therapeutic proteins (PPCA or beta gal) is targeted to neurons and glia by using cell-specific promoters. These transgenic models will be crossed with the respective null mice. This approach will allow us to assess the efficacy of neural cells to produce and secrete PPCA and beta-gal and of affected neighboring cells to take up the enzymes.
Aim 3. We will perform ex vivo gene transfer using genetically modified, deficient BM cells for syngeneic BMT of GS and GM1. This approach will create the most realistic scenario to gene therapy in the patients. In parallel, we will determine the efficacy of in vivo administration of a recombinant adeno-associated virus (rAAV) expressing the enzymes in correcting difficult to treat CNS regions. These combined studies should provide a solid base for the development of feasible cure for these diseases, leading to clinical treatments that have a greater chance of success and a lower risk of adverse effects.
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