Expression of transgenes in liver by gene therapy-mediated repopulation

A method that is often used to study liver biology and pathophysiology is to ectopically express a gene of interest in animal hepatocytes. The techniques most commonly used for gene expression, germline transgenesis and viral transduction, are limited by the magnitude of expense and screening work involved with transgenesis and the transience of expression with transduction. Previous studies found that fumaryl acetoacetate hydrolase (FAH) gene transfer to the liver cells of FAH-deficient mice leads to a selective repopulation by corrected cells to establish FAH expression in greater than 50% of total hepatocytes. I hypothesized that transgenes can be co-expressed with FAH to study liver biology. My goals were to (1) track liver repopulation in vivo, (2) control gene expression by RNA interference, and (3) study dominant human diseases. I successfully co-expressed various transgenes in the adult liver including the reporter luciferase, short hairpin (sh)RNA targeting luciferase, and both mutant and wildtype alleles of human alpha1-antitrypsin (hAAT). Mice injected with our FAH plus transgene co-expression constructs steadily increased in weight without drug therapy for their metabolic defect, an indication of normal liver function upon repopulation. Luciferase expression was visualized in vivo to monitor the kinetics of liver repopulation, and it expanded concomitant with correction of FAH deficiency. I found that shRNA could control the luciferase expression in vivo; the shRNA group had a 90% reduction in luciferase relative to the control, and the reduction persisted throughout liver repopulation. Finally, I co-expressed either mutant or wildtype hAAT and saw no difference in the ability of liver cells to repopulate. The wildtype protein could be detected in the serum but the mutant could not, indicating different processing of the protein. Furthermore, at 10 months post-injection the mutant protein accumulated in hepatocytes to develop the hispathological hallmark of disease, diastase-PAS positive hepatocyte inclusions. I conclude that transgenes linked to FAH will be stably expressed upon repopulation of the FAH-deficient mouse liver. This new method for gene expression can be applied to a wide variety of genetic and therapeutic studies of the liver.