Browsing by Subject "Mouse models"
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Item Identification of genes and pathways involved in Schwann cell tumor initiation, development and progression to develop novel drug targets(2013-06) Watson, Adrienne LeighIn 2012 in the United States alone, over 1.6 million people were diagnosed with cancer and nearly 600,000 people died as a result of cancer. Cancer is a disease in which inherited or acquired genetic changes endow cells with abnormal properties such as the ability to rapidly grow and proliferate, resist normal mechanisms of cell death and senescence, induce angiogenesis, invade surrounding tissues and eventually metastasize throughout the body. Malignant peripheral nerve sheath tumors (MPNSTs) are tumors composed of Schwann cells that have acquired these oncogenic characteristics. MPNSTs occur spontaneously in the general population at a rate of 1 in 100,000 people per year, but more commonly occur in the context of Neurofibromatosis Type 1 (NF1), an inherited genetic disease that occurs in 1 in 2,500 live births. NF1 patients develop neurofibromas, benign tumors derived from Schwann cells throughout the peripheral nerves of the body, due to loss of the tumor suppressor gene Neurofibromin 1 (NF1). Ten percent of patients with NF1 will incur additional genetic mutations in NF1 null cells that lead to the transformation of a benign neurofibroma into an MPNST. With little known about the genetic changes that cause sporadic MPNSTs or transformation from neurofibromas to MPNST, the current treatments for patients with MPNSTs are surgical resection and non-specific chemotherapy and the 5-year survival rate remains very low at less than 25%. In an attempt to better understand the genetic drivers of Schwann cell tumors and identify potential pathways that could be targeted by small molecule inhibitors, we conducted a Sleeping Beauty (SB) unbiased, forward genetic screen in mice. This screen uncovered hundreds of genes that may play a role in Schwann cell tumor initiation, development, progression and maintenance. The following thesis will describe in detail, several of the important findings that came out of the SB screen including: the discovery and validation of canonical Wnt/fÒ-catenin signaling as a pathway that plays a role in Schwann cell transformation, progression and tumor maintenance, and the profound clinical implications of co-targeting the MAPK and PI3K pathways, shown to be co-activated in the SB screen using small molecule, targeted therapies.Item Insights into determinants of cancer susceptibility, initiation, and progression:studies on medulloblastoma and Histiocytic Sarcoma in mouse models(2012-12) Been, Raha AllaeiThis dissertation presents a discussion of both perigestational dietary influence on cancer predisposition as well as somatic genetic determinants of cancer development. Both projects used genetically engineered mouse models of cancer. The introductory chapter gives a brief historical introduction to cancer, background information on models of cancer, and a short description of our current understanding of cancer. Chapter two presents data on how maternal diet can affect the risk for medulloblastoma in offspring. Medulloblastoma presents a dismal prognosis even for the patients who are successfully treated. Prevention strategies are therefore of great interest in addressing this disease. Chapter three discusses a mouse model of Histiocytic Sarcoma (HS) that was developed to identify genes that can contribute to initiation and cause progression of disease. The genetics of HS are not well understood. Our model could provide important information on molecular targets that can be used to treat this dreadful disease. The final, fourth chapter, provides a brief and broad overview of some of the major future likely sources of cancer control success with a focus on new research.Item Models and Gene Therapy for GM1-Gangliosidosis and Morquio Syndrome Type B(2018-12) Przybilla, MichaelGM1-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.