Winning Abstracts from the 2010 Medical Student Abstract Competition: Assessing Potential Toxicities Of Gene Therapy: Insertional Mutagenesis Risk Following Aav Vector-mediated Gene Transfer
Author: Hojun Li, University of Pennsylvania School of Medicine, Class of 2012
Hemophilia B is a severe bleeding disorder affecting 1 in 10,000 males. Gene therapy for Hemophilia B is a promising alternative to recombinant protein therapy. Long-term expression of coagulation factor IX (FIX) via stable gene transfer could reduce costs and risks associated with intravenous recombinant FIX infusions. In a human clinical trial (Manno, Nat Med 2006), our group has previously shown hepatic artery injection of an adenoassociated virus (AAV) vector expressing FIX (AAV-hFIX16) results in short-term efficacy and disease correction. A fundamental issue facing clinical gene transfer has to do with risk related to vector integration into the host genome. The consequences of AAV integration in in vivo systems is unknown. Here we present the results of a large-scale prospective study to determine whether integrated AAV vectors dysregulate nearby genes and increase cancer risk.
We performed a large-scale longitudinal study following 120 wild-type mice for 18 months after portal vein injection of either 5e12 vg/kg of AAV-hFIX16, 1e14 vg/kg of AAV-hFIX16, 1e14 vc/kg of empty AAV capsid, or saline. We measured expression of human FIX at 18 months by ELISA to confirm stable gene transfer. We determined the incidence of hepatocellular carcinoma (HCC) at 18 months post-injection and the incidence of HCC in 9 untreated mice of equivalent age. We cloned integrated vector copies using LM-PCR and mapped their genomic locations using 454 pyrosequencing. We performed microarray analysis on tumor tissue and adjacent normal tissue to determine if genes near integrated vector became dysregulated in their expression.
At 18 months we found circulating hFIX levels of 12 ug/mL in mice receiving 5e12 vg/kg, and 25 ug/mL in mice receiving 1e14 vg/kg, with no detectable hFIX in mice receiving empty capsid or saline. We found an HCC incidence rate of 9.1% in untreated mice, 0% in mice receiving saline or empty capsid, 3.8% in mice receiving 5e12 vg/kg, and 12.5% in mice receiving 1e14 vg/kg (p=0.64 comparing AAV treated to controls,), although the study was only powered to detect a 10% difference in HCC incidence. We cloned 251 unique integrant locations from tumor tissue. We found the expression changes of genes near integrants did not significantly change more than genes not near integrants (p=0.46).
Our large-scale prospective study of mice receiving AAV-hFIX16 demonstrated long-term stable gene transfer, but did not demonstrate a statistically significant difference in HCC incidence. When analyzing integrated vector in tumor tissue, we found that genes near these integrants were not any more dysregulated than genes not near integrants. These results suggest stable gene transfer of FIX is possible and that integrated AAV does not dysregulate nearby genes to promote oncogenesis.