Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy
Randy J. Chandler, … , Shawn M. Burgess, Charles P. Venditti
Randy J. Chandler, … , Shawn M. Burgess, Charles P. Venditti
Published January 20, 2015
Citation Information: J Clin Invest. 2015;125(2):870-880. https://doi.org/10.1172/JCI79213.
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Research Article

Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy

Abstract

The use of adeno-associated virus (AAV) as a gene therapy vector has been approved recently for clinical use and has demonstrated efficacy in a growing number of clinical trials. However, the safety of AAV as a vector has been challenged by a single study that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. Most studies have not noted genotoxicity following AAV-mediated gene delivery; therefore, the possibility that there is an association between AAV and HCC is controversial. Here, we performed a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the RNA imprinted and accumulated in nucleus (Rian) locus, and the resulting overexpression of proximal microRNAs and retrotransposon-like 1 (Rtl1) were associated with HCC. In addition, we demonstrated that the AAV vector dose, enhancer/promoter selection, and the timing of gene delivery are all critical factors for determining HCC incidence after AAV gene delivery. Together, our results define aspects of AAV-mediated gene therapy that influence genotoxicity and suggest that these features should be considered for design of both safer AAV vectors and gene therapy studies.

Authors

Randy J. Chandler, Matthew C. LaFave, Gaurav K. Varshney, Niraj S. Trivedi, Nuria Carrillo-Carrasco, Julien S. Senac, Weiwei Wu, Victoria Hoffmann, Abdel G. Elkahloun, Shawn M. Burgess, Charles P. Venditti

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Figure 1

Incidence of HCC in mice followed for 18 to 22 months after intrahepatic neonatal injection of AAV vectors.

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Incidence of HCC in mice followed for 18 to 22 months after intrahepatic...
(A) Schematic of AAV vectors packaged into AAV serotypes 2, 8, and 9 and used for gene delivery. AMBP, α-1-microglobulin; APOE, apolipoprotein E; ITR, AAV2 ITR; TBG, human TBG promoter. (B) Contribution of the murine background and transgene expression to the frequency of HCC. Untreated Mut+/– control mice (n = 51) were aged for 22 months and, at death, were assessed for hepatic carcinoma. Three HCCs were detected in this group. Mut+/– (n = 24) and Mut–/– (n = 24) mice were treated with 1 × 1011 to 2 × 1011 GC per pup of AAV8-CBA-Mut, and Mut+/– mice (n = 11) were treated with 1 × 1011 to 2 × 1011 GC per pup of AAV8-CBA-GFP. All untreated Mut–/– mice perished in the newborn period. (C) Relationship between AAV dose and the frequency of HCC following injection. Untreated Mut+/– control mice (n = 51) compared with Mut+/– and Mut–/– mice treated with AAV8-CBA-MUT at doses of 1 × 109–10 GC (n = 16) or 1 × 1011 to 2 × 1011 GC (n = 25) in the neonatal period. (See also Table 1.) *P < 0.01 (Fisher’s exact test, 2 tailed).