Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver

Abstract

Recombinant adeno-associated virus (rAAV) vector holds promise for gene therapy. Despite a low frequency of chromosomal integration of vector genomes, recent studies have raised concerns about the risk of rAAV integration because integration occurs preferentially in genes and accompanies chromosomal deletions, which may lead to loss-of-function insertional mutagenesis. Here, by analyzing 347 rAAV integrations in mice, we elucidate novel features of rAAV integration: the presence of hot spots for integration and a strong preference for integrating near gene regulatory sequences. The most prominent hot spot was a harmless chromosomal niche in the rRNA gene repeats, whereas nearly half of the integrations landed near transcription start sites or CpG islands, suggesting the possibility of activating flanking cellular disease genes by vector integration, similar to retroviral gain-of-function insertional mutagenesis. Possible cancer-related genes were hit by rAAV integration at a frequency of 3.5%. In addition, the information about chromosomal changes at 218 integration sites and 602 breakpoints of vector genomes have provided a clue to how vector terminal repeats and host chromosomal DNA are joined in the integration process. Thus, the present study provides new insights into the risk of rAAV-mediated insertional mutagenesis and the mechanisms of rAAV integration.

FIG. 1.

Distribution of integration sites in the rRNA gene repeats, the hottest spot for rAAV2 integration. One complete rRNA gene repeat is depicted. Ten independent integrations among 347 integrations isolated from in vivo-selected primary mouse hepatocytes were mapped in the rRNA gene repeats and are shown together in a rRNA gene repeat with closed arrows. Orientation of each arrow represents vector genome orientation relative to that for the transgene expression cassette. Most of the integrations landed on narrow regions containing the regulatory elements for the 45S pre-rRNA gene. Such elements include the enhancer repeats (Enh), spacer promoter (Sp-Pro), origin of bidirectional replication (OBR), amplification-promoting sequences (APS), and terminator (Term). These elements coincide with CpG islands. Open arrows show the 2 of 15 integration sites we previously isolated from nonselected mouse primary hepatocytes (21). Up-Term, upstream terminator.

FIG. 2.

Distribution of the breakpoints of the vector genome terminal of rAAV2 proviruses. A total of 571 breakpoints within the 250 nucleotides near the vector ends are shown with 5′ and 3′ breakpoints combined. A hot spot for recombination (nucleotide 76) bordering two distinct regions preferred and nonpreferred for recombination is shown on a flip-oriented ITR with secondary structure. A to D, subregions of the ITR.