Assessing the potential for AAV vector genotoxicity in a murine model

Abstract

IntroductionLiver-directed gene transfer using adeno-associated virus (AAV) vectors has the potential to serve as therapy for several inherited hematologic diseases. One such disease is the bleeding disorder hemophilia B, caused by a deficiency in coagulation factor IX (FIX). Currently, there are 2 clinical trials for hemophilia B that use liver-directed AAV-mediated gene transfer of the F9 gene (www. clinicaltrials.gov; identifiers NCT00515710 and NCT00979238). One of these trials reported transient efficacious circulating FIX levels (ϳ 10%) with the use of the vector AAV2-hFIX16. 1 Although AAV vectors are predominantly nonintegrating, with most of the transgene expression from stable episomes, 2 it has been shown through direct sequencing that integration can occur. 3,4 When integration takes place, there is a preference for integrating in regions where DNA breaks occur. These can be regions of endonuclease cleavage, 5 active transcription, 6-8 cytosine-phosphateguanosine (CpG) islands, 7,8 and palindromes. 9 All of these studies describing AAV vector genome integration identified vector integration sites through plasmid rescue of vectors containing bacterial origins of replication (ori). 4 Amplification of these plasmids in bacterial culture allows for sequencing of the integration junction between vector and host genome. Because of the bacterial selection involved in this method, bias may occur against recovering integrants whose size or sequence negatively affect bacterial growth, resulting in incomplete mapping of the full spectrum of integrants.Vector genome integration has been associated with adverse events; integrating ␥-retroviral vectors were implicated in the clonal expansion of transduced cells in 3 clinical studies, 2 for X-linked severe combined immunodeficiency 10,11 and the other for chronic granulomatous disease. 12,13 Although AAV vectors integrate at a much lower frequency than retroviral vectors, low-level AAV vector integration in transduced cells may still be a concern. A compelling argument supporting low genotoxic risk of AAV vectors comes from long-term follow-up of liver-directed AAVmediated gene transfer in canine and murine models. Of 77 dogs receiving AAV vector at doses up to 3.4 ϫ 10 12 vector genomes(vg)/kg and followed for Յ 10 years, none developed liver tumors as assayed by ultrasound, computed tomographic (CT) scan, and magnetic resonance imaging (MRI) 14,15 (K.A.H., V.R.A., and Timothy C. Nichols, unpublished data, October 15, 2010). Similarly, Ͼ 300 mice receiving AAV vectors with a therapeutic transgene at doses up to 4 ϫ 10 13 vg/kg and followed Յ 14 months have not shown a difference in tumor incidence compared with control mice. 16,17 However, a study by one group reported an increase in tumor incidence that was attributed to AAV vectors. 18 These investigators reported that administration of an AAV serotype 2 (AAV2) vector encoding ␤-glucuronidase in neonatal mice resulted in a significant increase in incidence of hepatocellular carcinoma (HCC), a tumor commonly fou...

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

“…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. Nevertheless, this study also used integration profiling and gene expression analysis to demonstrate that the HCCs appeared free of clonal integration events, or gene expression changes related to rAAV integration.…”

“…Donsante et al injected the mice on postnatal day (PND) 1-2; Bell et al and Li et al injected young adult animals. 39,45,46 These data suggest that mice injected during the newborn period may be more susceptible to the development of rAAV-associated HCC and, furthermore, that the HCC association was not mouse model dependent.…”

“…Nevertheless, this study also used integration profiling and gene expression analysis to demonstrate that the HCCs appeared free of clonal integration events, or gene expression changes related to rAAV integration. 46 A separate study showed that hepatic tumors were observed in young adult mice following intravenous injection of a high dose (2 · 10 10 vp/mouse) of an rAAV vector expressing a factor IX cDNA containing the Padua mutation, but occurred at an insignificant rate of *15%. Genomic characterization was not performed on the HCC in this study.…”

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).…”