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.

Absence of ocular malignant transformation after sub-retinal delivery of rAAV2/2 or integrating lentiviral vectors in p53-deficient mice

“…rAAV integration studies in the mouse liver reported that a high frequency (53-72%) of integration events occurred in transcribed regions. 9,10 A number of integration hot-spots in the mouse genome was identified, the most prominent being rRNA gene repeats. Those studies also identified integrations (3.5%) near or within cancer-related genes.…”

Adeno-associated virus integration: virus versus vector

“…In an expanded study employing the HT1 mouse model and in vivo selection of hepatocytes bearing rAAV integrants, Nakai et al 26 injected HT1 mice with 3 Â 10 11 rAAV serotype 2 particles encoding the FAH gene. Libraries of integrated proviral sequences were constructed from liver DNA isolated from mice that had begun the in vivo selection process at either 3 or 6 weeks post-infusion.…”

Existence of transient functional double-stranded DNA intermediates during recombinant AAV transduction

“…Circular DNAs were eventually converted into high-molecular weight concatemers and were presumably stabilized. There are two major categories of stabilized AAV genomes that can be detected months after rAAV transduction: extrachromosomal concatemers and integrated AAV genomes (29,30). Although wild-type AAV frequently underwent site-specific integration into human chromosome 19, similar integrations by rAAV were infrequent (31,32).…”