216. Long Term Portal Vein Administration of AAV-WPRE Vector Results in Increased Incidence of Neoplastic Disease and Hepatic Pathology
Authors: Jennifer E. Embury, Catherine C. Charron, Amy E. Poirier, Andreas Zori, Russ Carmichael, Terry R. Flotte, Philip J. Laipis
Journal: Molecular Therapy (2006)

Utilizing the Pahenu2 mouse model for phenylketonuria, we developed several expression vectors containing the Woodchuck Hepatitis Virus post-transcriptional regulatory element added in a rAAV-mPAH construct (rAAV-mPAH-WPRE). While these modifications resulted in increased efficacy in reducing hyperphenylalanemia, we observed severe hepatic pathology and increased incidence of hepatic and systemic neoplastic disease at the termination of these studies. Group 1 received AAV- CBmPAHdimer-WPRE with doses ranging from 1.5x109 to 4e10 IU and was sacrificed at 11 months. 44% (4/9) developed neoplastic disease that included 2 hepatocellular carcinomas, 1 hepatoma, and 1 bronchiogenic carcinoma. 6/9 (66%) had severe hepatic morphology which included myriad cytoplasmic inclusion bodies (1/9) and hepatocellular metaphase arrest. Group 2 was sacrificed at 8 months and had received a similar dose range and vector as group 1 but was ovariectomized and received either a testosterone or placebo pellet. 46% (6/13) developed the most unusual and poorly differentiated forms of neoplasia which included a peritoneal carcinosarcoma, a mesothelioma-like neoplasm of the pleura and epicardium, bilateral renal carcinoma, pulmonary and splenic lymphosarcoma, and two blastoma-like tumors with hepatic sinusoidal involvement. 7/13 (54%) also had pathologic changes in the liver as in group 1. Experimental group 3 was sacrificed at 6 months and received 4e9 AAV-CB-mPAHhd-WPRE as well as 4x109 to 2x1010 AAV-CB-RzI209 vector. None developed neoplastic disease but 60% (3/5) did develop altered hepatocellular morphology as above. Mice from group 4 were sacrificed at 6 and 12 months and received between 4e9 and 3e10 IU of AAV-CB-mPAHhrd+RzI209 (no WPRE). None of these mice (0/8) developed neoplastic disease, but 37.5% (3/8) developed mild to moderate changes in hepatocellular morphology that were consistent with increased metabolic activity. The woodchuck hepatitis virus (WHV) contains a highly conserved 154 amino acid HBV X protein (HBx) that is implicated as a key element in the development of hepatocellular carcinoma. HBx is a trans- activating protein that is often expressed in hepatocellular carcinoma (HCC) and surrounding nontumor liver tissue. The WPRE element we used included an amino terminal 60 amino acid fragment of the HBx protein (1503-1680). To determine if vector was present in neoplastic tissue, and more specifically to look for the presence of HBx, PCR (TAQMan©) was employed using HBx probes. HBx DNA was not detected in tumoral tissue although it was present in transduced liver. Immunohistochemical detection of Hbx antigen was observed on the periphery of 1 liver tumor and scattered randomly throughout an intestinal tumor. These results imply that AAV-vector is not integrated into the tumor, but rather it is possible the Hbx element is acting in a "hit and run" fashion when administered systemically and may activate cellular oncogenes at random.