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.

Figure 5. Regulatory elements influence HCC incidence after AAV gene delivery.

(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.

Figure 4. Global RNA and miRNA expression profiles of HCC versus normal liver tissue.

(A) Dendrogram displays the hierarchical clustering of genes with similar expression patterns. Red indicates an increase and blue indicates a decrease in gene expression in the HCC versus normal liver tissue. The scale (red to blue) represents the ratios of differential expression based on signal intensity, while the numbers to the right and left denote the Euclidean sample dissimilarity (Partek Genomic Suite). (B) RNA expression changes of 5-fold or greater in HCCs (n = 29) relative to normal liver tissue (n = 28) detected with RNA microarray. (C) miRNA expression changes of 5-fold or greater in HCCs (n = 12) relative to normal liver tissue (n = 6) detected with miRNA-specific microarray. P < 0.01 for all changes in expression (ANOVA). (See also Supplemental Tables 3–5.)

Figure 3. Vector integrations in AAV-associated HCCs.

(A) Genomic locations (UCSC Genome Browser, Genome Reference Consortium Mouse Build 38 December 2012, mm10) of HCC integration junctions validated after PCR amplification and Sanger sequencing compared with HCC integration junctions identified previously by Donsante et al (9). (See also Supplemental Tables 1 and 2.) The bolded gene, Rtl1, and microRNAs were increased in expression in the HCCs. (B) Within the Rian locus, semiquantitative targeted PCR amplification of common AAV vector-Mir341 junctions in both HCCs and adjacent normal livers from the same mice and mice (C1–C3) that were treated with AAV but did not develop cancer. The numbers above the HCC lanes refer to the HCC integration junctions presented in A. Comparison to vector amplification standards (left) suggests the integration junctions are present an average of approximately 1 copy per hepatocyte.

Figure 2. Vector integration profile in normal livers and HCCs after AAV delivery.

(A) Genes with 3 or more independent AAV integrations identified by integration capture and subsequent high-throughput sequencing in HCCs (n = 33) in comparison to integrations in normal livers (n = 31). The fragment count is equal to the number of times a unique integration sequence is detected. Due to size constraints, fragment count ≥1 shows all genes with 3 or more unique integrations. Fragment counts ≥100 are filtered for genes with more strongly amplified integration sites. *P < 0.01 (Fisher’s exact test, 2 tailed). (B) Integrations of AAV8-CBA-Mut, AAV8-CBA-MUT, and AAV8-TBG-Mut vectors into the albumin (red), α-fetoprotein (blue), and Rian (green) loci. Each gene is depicted with exons in solid black, with solid lines representing an independent integration event. Thicker lines indicate multiple AAV integrations. The thick line over the Rian locus is created by the 12 independent HCC-associated integrations that map within a small genomic window. (See also Supplemental Table 1.)

Figure 1. Incidence of HCC in mice followed for 18 to 22 months after intrahepatic neonatal injection of AAV vectors.

(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 × 10^9–10 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).