Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy
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
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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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 5

Regulatory elements influence HCC incidence after AAV gene delivery.

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Regulatory elements influence HCC incidence after AAV gene delivery. (A)...
(A) Frequency of HCC in mice when treated with different AAV serotypes and enhancer promoter elements in comparison to that of uninjected Mut+/– control mice (n = 51). Mut+/– mice treated with AAV8-CBA-Mut (n = 24), AAV8-TBG-Mut (n = 11), and AAV2-CBA-Mut (n = 11) at a dose of 1 × 1011 to 2 × 1011 GC per pup, AAV9-CBA-Mut (n = 12) at a dose of 1 × 1010 GC per pup, and AAV8-hAAT-synMUT at a dose of 1 × 1011 GC per pup (n = 10, 8 Mut+/– and 2 Mut+/+ mice). (See also Table 1.) *P < 0.01 (Fisher’s exact test, 2 tailed). (B) Livers from mice (n = 5; 3 Mut+/– and 2 Mut+/+ mice) treated with AAV8-hAAT-synMUT after direct hepatic injection were used to characterize vector integrations. The fragment count is equal to the number of times a unique integration sequence is detected. Fragment count ≥1 shows genes with the greatest number of unique integrations. Fragment counts ≥100 are filtered for genes with more strongly amplified integration sites. (See also Supplemental Table 6.) (C) Six unique AAV integrations detected at 18 months of age in the normal appearing livers of 3 of 5 treated mice. (D) Schematic of hypothesis. CBA and TBG, but not the hAAT promoter enhancer, are capable of promoting increased transcription of proximal genes (transactivation) that drive the formation of HCC. The orientation of the vector integration events was not determined and therefore remains hypothetical.