No Evidence for Tumorigenesis of AAV Vectors in a Large-Scale Study in Mice

Abstract

Six hundred ninety-five mice received adeno-associated virus (AAV) vectors, mostly via portal vein injection. At necropsy, the livers were inspected for tumors, and tissue sections were prepared for histology. We observed only one tumor, a lipoma, resulting in a tumor frequency of 0.14%. This tumor contained fewer vector genomes per total DNA than the surrounding liver tissue, as shown by quantitative PCR. In another mouse we found a macroscopically visible nodule containing lymphocytes. Immunohistochemistry revealed cells not of monoclonal origin, and they contained fewer AAV genomes than the surrounding hepatocytes. There were no macroscopic tumors in 226 control mice. Upon microscopic examination, lymphocytic infiltrates were found in 5% of livers of both control and vector-treated mice; no transgene expression was seen in those infiltrates in AAV-injected animals. Compared to an average frequency of spontaneous liver tumors in C57BL/6 mice (0-10%), and given the absence of high levels of vector DNA in the observed tumor, we conclude that AAV vectors do not predispose these target animals to the formation of liver tumors.

Induction of hepatocellular carcinoma by in vivo gene targeting

“…Although insertion of an entire promoter/enhancer would be an unusual type of spontaneous mutation, epigenetic dysregulation could have produced the human tumors, because we observed reduced cytosine methylation within a CpG island adjacent to the syntenic insertion site in some C3 HCCs. Furthermore, our findings raise the possibility that unintended vector integration at the human locus might also lead to C3 HCC in gene therapy trials, despite numerous preclinical studies demonstrating safety in other animal models (8)(9)(10)43).…”

“…A later study of sleeping beauty transposition also found HCCs with integrations at this locus (7). Both studies highlight the potential genotoxicity of vector integration in hepatocytes, but their significance remains controversial because other reports have shown that animals do not develop HCC after AAV vector injections (8)(9)(10). The integration site locus contains a complex set of imprinted genes that are uniquely dysregulated after reprogramming to pluripotency (11), and two noncoding RNAs (Rian and Mirg) that contain multiple snoRNAs and microRNAs (12).…”

Administration-route and gender-independent long-term therapeutic correction of phenylketonuria (PKU) in a mouse model by recombinant adeno-associated virus 8 pseudotyped vector-mediated gene transfer

“…14, 25 We thus concluded that AAV serotype 8 is superior to serotype 2 or 5 vectors for liver transduction, and was efficient for gene transfer to correct liver PAH (Figure 4d). Although no evidence for tumorigenesis of AAV vectors in mice was found, 38 a potential oncogenic activity of the WPRE sequence upon recombinant viral therapy containing this DNA element was reported. 39 We performed with a limited number of our experimental mice (n ¼ 3) a careful inspection for tumors, including histological analysis of liver.…”

Recombinant Adeno-Associated Viral Integration and Genotoxicity: Insights from Animal Models

“…In 2005, a large retrospective study of mice treated with rAAV did not find an association between rAAV treatment and HCC in mice. 45 A second, relatively large study, involving 132 mice, specifically designed to address the issue of HCC following intravenous administration of rAAV vectors, showed no statistically significant increase in HCC in mice injected with an rAAV vector encoding clotting factor IX. 46 However, the small number of HCCs observed in this study, five in total, four of which occurred in the rAAV-treated group, may not have been sufficient to detect a significant association.…”