March 2012

Document Type


Degree Name



Dept. of Cell and Developmental Biology


Oregon Health & Science University


Many inborn errors of metabolism are caused by deficiencies in hepatic enzymes, thereby making whole orthotopic liver transplantation an effective therapy for these conditions. However, few patients benefit from or even undergo this procedure due to the limited availability of donor organs, the high cost and significant morbidity and mortality associated with the procedure, as well as the long-term requirement for immune suppression. Most pediatric metabolic diseases are caused by a specific loss-of-function in only hepatocytes with otherwise normal liver anatomical structure. This is in contrast to many adult liver disorders treated with whole liver transplant where severe anatomical abnormalities have accumulated that obstruct proper liver function. Thus, many pediatric patients with metabolic disease have little need for whole organ transplant and would benefit from restoration of enzymatic function through gene therapeutic approaches or hepatocyte replacement via cell transplantation. My thesis attempts to address both of these potential therapeutic strategies. In Chapter 2, I provide the first proof-of-principle data establishing that recombinant adeno-associated viral (rAAV) vectors are capable of functional correction of a metabolic liver disease in vivo through a gene targeting approach. This work was the first to demonstrate the stability of gene targeting in both neonatal and adult mice using two different pseudotyped rAAV serotypes, rAAV2/2 and rAAV2/8. Furthermore, this work established the functional time course for correction using both serotypes of rAAV at a range of viral doses. In Chapter 3, I expand upon my established rAAV gene targeting paradigm from Chapter 2 and demonstrate increased gene targeting capabilities through transient use of the pharmacologics vanillin or bortezomib. The natural product vanillin acts as a potent inhibitor of the undesired repair pathway nonhomologous end-joining (NHEJ) by inhibiting the protein kinase DNA-PK. Bortezomib is a proteasomal inhibitor that prevents AAV capsid degradation. Broadly, this study was the first to show the potential clinical benefit of inhibiting a non-desired DNA repair pathway for increasing AAV-mediated gene targeting with an animal model of any disease in a clinically relevant manner. More specifically, this work was the first to show the benefits of transient NHEJ inhibition with vanillin at improving AAV mediated gene targeting for an in vivo mouse model of hereditary tyrosinemia type I (HTI) to near therapeutic levels. In Chapter 4, I describe the use of a small molecule agent to transiently apply positive selection for transplanted hepatocytes. Most liver diseases lack the advantage of positive selection, thus the ability to transiently apply positive selection for correction of liver disorders would represent a major advance. The small molecule 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate (CEHPOBA) was tested both in vitro and in vivo for its ability to inhibit target pathways. As proof of principle, we attempted to positively select hepatocytes genetically resistant to CEHPOBA by transplanting into wild-type recipients along with CEHPOBA treatment. Time course analyses with 3-5 week courses of CEHPOBA following transplantation showed a linear relationship between treatment length and degree of repopulation. Compared to controls, CEHPOBA-treated recipients had 10-100 fold increases in liver repopulation. This work established that selective repopulation in a non-selective wild-type liver through transplant of genetically resistant hepatocytes and daily administration of the pharmacological inhibitor CEHPOBA could provide transient selection to donor hepatocytes in vivo. Based on these findings, it is thought that the combination of transient shRNA knockdown of upstream enzymatic targets in donor cells, hepatocyte transplantation and drug treatment with CEHPOBA could constitute a new path for future therapeutic liver repopulation.




School of Medicine



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