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Title: Hepatitis virus protein X-Phenylalanine Hydroxylase fusion proteins identified in PKU mice treated with AAV-WPRE vectors

item EMBURY, JENNIFER - University Of Florida
item FROST, SUSAN - University Of Florida
item CHARRON, CATHERINE - Imperial College
item MADRIGAL, EMILIO - University Of Florida
item Perera, Omaththage
item POIRIER, AMY - University Of Florida
item ZORI, ANDREAS - University Of Florida
item CARMICALR, RUSS - University Of Florida
item FLOTTE, TERRENCE - University Of Florida
item LAIPIS, PHILIP - University Of Florida

Submitted to: Gene Therapy and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/5/2008
Publication Date: 6/6/2008
Citation: Embury, J.E., Frost, S., Charron, C.E., Madrigal, E., Perera, O.P., Poirier, A.E., Zori, A., Carmicalr, R., Flotte, T.R., Laipis, P.J. 2008. Hepatitis virus protein X-Phenylalanine Hydroxylase fusion proteins identified in PKU mice treated with AAV-WPRE vectors . Gene Therapy and Molecular Biology. 12:69-76.

Interpretive Summary: A gene therapy expression vector that included a part of a regulatory sequence of the Woodchuck hepatitis virus was used for treatment of phenylketonuria (PKU) in a mutant mouse model. Unintentional abnormal cell growth in various tissues and liver disease were observed in 44% and 70% of treated mice, respectively. The gene therapy vector contained a part of Woodchuck hepatitis virus "X-protein" that is known to induce tumor formation. This X-protein was found to form a fusion protein with the phenylalanine hydroxylase enzyme used to treat PKU and may have been responsible for causing unusually high rates of cancer and liver disease in treated mice.

Technical Abstract: Utilizing the Pahenu2 mouse model for phenylketonuria (PKU), we developed an improved expression vector containing the Woodchuck Hepatitis Virus post-transcriptional regulatory element inserted into a rAAV-mPAH construct (rAAV-mPAH-WPRE) for treatment of PKU. Following portal vein delivery of these vectors to Pahenu2 mice, we observed the unintentional development of neoplastic disease (44%) and hepatic pathology (70%) in WPRE-treated mice. Our vector contained a portion of the oncogenic hepadnoviral “X-protein” in the WPRE segment that had been intentionally modified in an attempt to prevent its expression. The hepadnoviral X- protein encoding sequence is known to function as a mediator in oncogenic activity (Murakami, 1999). We have evidence that the X-protein fragment unexpectedly formed a fusion protein with a phenylalanine hydroxylase transgene in our vector and suspect this fusion protein may have been responsible for the high rate of unusual types of cancer and hepatic pathology. These results are not to imply that the use of the WPRE element will always result in the development of cancer. But in this particular instance, an unanticipated event may have ensued when the X-protein formed a fusion protein with the transgene. This is a cautionary illustration to be considered when developing genetic therapies to treat diseases.