Nowadays, recombinant adeno-associated viruses (rAAV) constitute one of the most successful vectors for human gene therapy. These viral vectors allow a long-term transgene expression mainly due to their ability to form stable concatemeric DNA structures. Interestingly, although it is a rare event, rAAV have been shown to randomly integrate into the host genome.The inverted terminal repeats (ITR) have been shown to be preferred breakage points within the rAAV vectors. Current analysis of AAV integration are based on the linear amplification mediated (LAM)-PCR technology, here the genomic regions adjacent to the ITRs are amplified and sequenced for the determination of the integration sites (IS). However, recent studies have shown that vector breakage may also occur along the whole vector sequence, thus requiring the development of new methods able to identify the integration of internal vector regions.We present a novel method for the detection of rAAV integration based on the LAM-PCR technology by employing simultaneously five primer sets covering the whole AAV vector sequence. This multiplex LAM-PCR was used, in parallel with the standard 5’ LAM-PCR, in liver, spleen and adrenal gland biopsies from monkeys injected with 1×1013 viral genomes (vg)/kg or 5×1013 vg/kg of the AAV2/5-AAT-coPBGD vector (this vector has been used in a phase I clinical trial for acute intermittent porphyria). Subsequent 454 sequencing and data analysis of ≈1 million raw sequences for each tissue allowed the identification of near 800 integration sites (IS). As it has been previously reported, vector integration occurs within different regions of the ITR, although preferentially in the C-region. In addition, multiplex LAM-PCR showed breakage at different positions of the AAV vector genome in all the organs analyzed.Our data show the increased sensitivity of this method and its suitability for the analysis of viral integration occurring at inner regions of the rAAV vector. Thus, this new method will provide a deeper insight into the interaction of AAV vectors with the host genome and will contribute to increase the thoroughness of the safety studies for the increasing number of gene therapy clinical trials using rAAV vectors.
