Przybilla, Michael2021-02-222021-02-222018-12https://hdl.handle.net/11299/218689University of Minnesota Ph.D. dissertation. December 2018. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor: Chester Whitley. 1 computer file (PDF); xiii, 172 pages.GM1-gangliosidosis and Morquio syndrome type B are lysosomal diseases caused by deficiencies in the lysosomal enzyme β-galactosidase (β-gal). β-gal is responsible for catabolizing the terminal β-linked galactose residues in GM1 and GA1 ganglioside, keratan sulfate, and oligosaccharides. If β-gal enzyme activity is deficient, these macromolecules accumulate within the lysosomes, resulting in either severe neurodegeneration in GM1-gangliosidosis or severe skeletal dysplasia in Morquio syndrome type B. Sadly, no therapies for these debilitating diseases exist, so the development of novel treatments is of the utmost importance; however, to be able to test these new treatments, animal models are necessary. Previous murine models of GM1-gangliosidosis were generated using an inefficient method to disrupt the Glb1 gene by introducing foreign DNA into the coding sequence. While useful, these mutations do not recapitulate those that could be found in patients with GM1-gangliosidosis. Utilizing CRISPR-Cas9 genome editing, the mouse β-gal encoding gene was targeted to generate mutations that resulted in two novel mouse models of β-gal deficiencies (Chapter II). In one line, a 20 bp deletion was generated to remove the catalytic nucleophile of the β-gal enzyme, resulting in a mouse devoid of β-gal enzyme activity (β-gal-/-). This resulted in ganglioside accumulation and severe cellular vacuolation throughout the central nervous system (CNS). β-gal-/- mice also displayed severe neuromotor and neurocognitive dysfunction, and as the disease progressed, the mice became emaciated and succumbed to the disease by 10 months of age (Chapter III). Overall, this model phenotypically resembles a patient with infantile GM1-gangliosidosis. In the second model, a missense mutation commonly found in patients with Morquio syndrome type B, GLB1W273L, was introduced into the mouse Glb1 gene (Glb1W274L). Mice harboring this mutation showed a significant reduction in β-gal enzyme activity (8.4-13.3% of wildtype) but displayed no marked phenotype after one year of observation (Chapter IV). This is the first description of using CRISPR-Cas9 genome editing to generate mouse models of a lysosomal disease. With these models in hand, preliminary experiments were conducted to test the functionality of a novel gene therapy to treat these diseases (Chapter V). Previous studies in lysosomal diseases have shown that tissue-specific expression of lysosomal enzyme ameliorates the disease pathology, including improvement of neurocognitive function. Here, a gene therapy system was designed to integrate the human GLB1 cDNA into the albumin locus by creating a double-strand break in the DNA by an AAV8-encoded nuclease. Theoretically, this integration of GLB1 cDNA would be achieved by co-injecting a second AAV8 vector encoding the transgene that is flanked by homologous sequence to the albumin locus, allowing for homology directed repair to incorporate the sequence. 30 days post treatment, plasma enzyme activity was 4.8-fold higher than heterozygous levels. However, by four months post-treatment, β-gal enzyme activity in plasma from treated β-gal-/- mice decreased to heterozygous levels. Four months following injection, β-gal enzyme activity in a subset of treated β-gal-/- mice was observed in the liver and spleen. Motor function testing on the rotarod showed that the amount of enzyme being produced does not prevent the neurological symptoms of the disease. This preliminary data shows that this gene therapy system can produce functional β-gal enzyme that is secreted into the plasma and is capable of being taken up into peripheral tissue. Future studies focused on optimizing the dose of AAV to provide a higher enzyme level will be important for the success of this therapy for β-galactosidase deficiencies.enCRISPR-Cas9GM1-gangliosidosisLysosomal diseaseMorquio syndrome type BMouse modelsThesis or Dissertation