Recombinant adeno-associated virus integration sites in murine liver after ornithine transcarbamylase gene correction

Abstract

Recombinant adeno-associated viruses (rAAVs) have been tested in humans and other large mammals without adverse events. However, one study of mucopolysaccharidosis VII correction in mice showed repeated integration of rAAV in cells from hepatocellular carcinoma (HCC) in the Dlk1-Dio3 locus, suggesting possible insertional mutagenesis. In contrast, another study found no association of rAAV integration with HCC, raising questions about the generality of associations between liver transformation and integration at Dlk1-Dio3. Here we report that in rAAV-treated ornithine transcarbamylase (Otc)-deficient mice, four examples of integration sites in Dlk1-Dio3 could be detected in specimens from liver nodule/tumors, confirming previous studies of rAAV integration in the Dlk1-Dio3 locus in the setting of another murine model of metabolic disease. In one case, the integrated vector was verified to be present at about one copy per cell, consistent with clonal expansion. Another verified integration site in liver nodule/tumor tissue near the Tax1bp1 gene was also detected at about one copy per cell. The Dlk1-Dio3 region has also been implicated in human HCC and so warrants careful monitoring in ongoing human clinical trials with rAAV vectors.

FIG. 1.

Flow chart summarizing the analysis of rAAV vector integration site distributions. Initially, 10 samples were selected from the study of Bell et al. (2006), consisting of paired tumor and normal liver DNA specimens from five mice containing liver nodules or tumors after exposure to rAAV. These were used to generate 10 integration site libraries, one per specimen, that were sequenced using the 454/Roche pyrosequencing method, yielding 1,580 unique integration site positions. Of these, 11 were selected for verification because of possible involvement in transformation based on high relative abundance (n=8), proximity to cancer-related genes (n=3), or location in the Dlk1–Dio3 locus (n=5), which was previously suggested to be associated with rAAV integration in hepatocellular carcinoma. Note that the sum of the numbers above is greater than 11 because some sites were chosen based on more than one criteria (see Table 1). Of the 11 integration sites, 9 could be validated by PCR amplification, cloning in bacterial plasmids, and DNA sequencing. Of these, two out of nine were present at ∼1 copy per cell (Table 1). rAAV, recombinant adeno-associated virus; PCR, polymerase chain reaction.

FIG. 2.

Distribution of rAAV integration sites in liver nodule/tumor and flanking normal tissue for the five mice studied. For each mouse, the chromosomes are shown numbered sequentially in circular ideograms (outermost ring). Progressing inward, relative gene density is shown by the red bars, positions of integration sites in normal liver is shown by the green dots, and integration sites in nodule/tumor samples is shown by the brown dots.

FIG. 3.

Integration sites of rAAV in tumor/nodule samples at the (A) Dlk1–Dio3 and (B) Tax1bp1 loci. Integration sites are shown as bright green vertical lines, and the mouse of origin is shown to the left. The integration site positions reported by Donsante et al. (2007) are shown in (A) by the blue-green lines. The snoRNA annotation (blue lines) was derived from the annotation of the syntenic region of the human genome, and the miRNA annotation (red lines) was from experimental data in mice. All annotation was adapted from tables at http://genome.ucsc.edu/.