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The role of human genetic variabilty in response to pharmaceuticals or xenobiotics (commonly referred to as pharmacogenomics and toxicogenomics, respectively) is a continually-expanding field of study. However, the ability to study the function of a single gene under normal or disease states is hampered by the large interindividual variability found in the human population. To control for environmental and genetic effects, we use inbred genetically-engineered mice to study human gene function in vivo. Through gene targeting in embryonic stem cells and various transgenic methodologies, it is possible to develop mice with specific gene deletions and/or express any chosen gene of interest in a defined temporal and spacial pattern. In addition to the use of the murine stem cells for developing "knockout" mice, these cells can be differentiated in vitro to any of the three germ layer lineages (ectoderm, endoderm and mesoderm). We are exploiting this feature using human embryonic stem cells in collaboration with members of ISCRM (Institute for Stem Cells and Regenerative Medicine) here at UW. Current projects under investigation in my laboratory include:
1. Development of a "humanized" Cyp4b1 knockout mouse to study the physiological role of this gene and it's function in endobiotic/xenobiotic metabolism. CYP4B1 cloned and expressed from a variety of different species bioactivates arylamine xenobiotics and may play a role in oxidation of endogenous fatty acids such as arachidonic acid and the leukotrienes. The functional activity of the human form however, is unknown due to the inability to express the gene in vitro. Expressing human CYP4B1 in a Cyp4b1 knockout mouse will address the endogenous function of the murine gene and functionality of the human enzyme.
2. Function of "orphan" CYP4 cells in health and disease. Two different members of the CYP4 family have been recently linked to seemingly disparate diseases, Lamellar Ichthyosis and Bietti's Crystalline Dystrophy (CYP4F22 and CYP4V2, respectively). Using a combination of patient-derived cell lines, engineered cells of target tissues, and a mouse knockout model, we are dissecting the biochemical functions(s) of CYP4V2 in fatty acid homeostasis. Future work using these same approaches will be applied toward CYP4F22. We say these two diseases are only "seemingly" disparate because another rare genetic condition, Refsum's disease, affects lipid biochemistry in both the eye and skin, resulting in a pathophysiological state mimicking a combined disruption of CYP4V2 and CYP4F22 function.
3. Utility of embryonic stem (ES) cells as a source of human hepatocytes.
There have been recent advances in the ability to differentiate ES cells into a variety of specific cell fates including oligodendrocytes, cardiomyocytes and hepatocytes. We are working on developing optimal conditions for hepatocyte differentiation using both NIH-approved ES cell lines, as well as induced pluripotent stem cell lines (iPSCs). Human hepatocytes are a key component of preclinical drug development because animal models are not always capable of fully replicating what occurs in people. In addition, idiosyncratic hepatotoxicity is one of the main reasons for FDA withdrawl of drug approval. Because human hepatocytes are a finite resource, with no consistent quality control or genetic profile, the ability to differentiate hepatocytes from stem cells is highly desirable. Given that CYP450 function is a hallmark of liver function, we are defining our approaches based on the ability to express metabolically active enzymes.
Dr. Kelly earned his Ph.D. in Biochemistry from the University of Washington in 1996. Following a post-doctoral fellowship in molecular toxicology in the Department of Environmental Health at the University of Washington, he worked at Targeted Genetics Corporation evaluating the safety and efficacy of gene therapies. He has served as the manager of the Center for DNA Sequencing and Gene Analysis in the School of Pharmacy since 2003, and was appointed to the position of Research Assistant Professor in April 2008.
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