Transplantation of genetically modified hepatocytes containing a copper-transporting ATPase, direct hepatic delivery, and the potential for the treatment of Wilson's disease

Wilson's disease is caused by a single gene defect in a copper transporting ATPase. The ATP7b protein, expressed primarily in the liver, is non-functional in patients due to a mutation in the gene sequence. Patients with Wilson's disease typically present with increased copper concentrations, chronic hepatitis, and fulminant liver failure. The murine Wilson's Disease animal model is the ATP7b mouse strain. If viable hepatocytes can be extracted from donor rodents, and then be found in recipient rodents after intrasplenic injection, then the introduction of a cDNA encoding a functional ATP7b protein could perhaps help lower copper concentrations in recipient rodents afflicted with hepatic copper accumulation. Primary hepatocytes were isolated using retrograde perfusion, labeled, injected into recipient spleens, followed over a period of time, and then identified within the recipient liver and spleen. Preliminary observations suggested that intrasplenic transplantation of hepatocytes cause a consistent series of pathophysiological events, and can be found in the liver and spleen for several weeks. The RS-VPCR control vector was created by placing a control 500 base pair insert within the pcDNA 4/HisMax-TOPO plasmid. Numerous ratios of lipid to plasmid were tested to determine the highest rate of transfection. Recipient mice received an intrasplenic injection of syngeneic derived donor hepatocytes, transfected with the control insert sequence plasmid. PCR confirmed the presence of the control DNA sequence in the liver and spleen of the recipient animals at 1 and 2 days post injection. The ATPase gene was subcloned into a pcDNA 4/HisMax-TOPO plasmid and then transfected into hepatocytes, for intrasplenic transplant. Samples for all time points were negative for the ATP7b insert by PCR. As an alternate route, direct hepatic injection was conducted using cationic lipid and the ATP7b plasmid. At 7, 14, 21, and 28 days post direct hepatic injection, livers and spleens were collected for DNA, RNA and protein isolation, and blood was collected for serum copper analysis. DNA, RNA and proteins specific for the ATP7b insert were predominantly found in all samples for all time points. However, copper concentrations did not show a large significant change. Additional studies with enhanced protein expression could provide more information to allow improvement in copper concentrations in this Wilson's disease animal model.