title: Integration frequency and intermolecular recombination of rAAV vectors in non-human primate skeletal muscle and liver.
abstract: The comprehensive characterization of recombinant adeno-associated viral (rAAV) integration frequency and persistence for assessing rAAV vector biosafety in gene therapy is severely limited due to the predominance of episomal rAAV vector genomes maintained in vivo. Introducing rAAV insertional standards (rAIS), we show that linear amplification-mediated (LAM)-PCR and deep sequencing can be used for validated measurement of rAAV integration frequencies. Integration of rAAV2/1 or rAAV2/8, following intramuscular (IM) or regional intravenous (RI) administration of therapeutically relevant vector doses in nine adult non-human primates (NHP), occurs at low frequency between 10(-4) and 10(-5) both in NHP liver and muscle, but with no preference for specific genomic loci. High resolution mapping of inverted terminal repeat (ITR) breakpoints in concatemeric and integrated vector genomes reveals distinct vector recombination hotspots, including large deletions of up to 3 kb. Moreover, retrieval of integrated rAAV genomes indicated approximately threefold increase in liver compared to muscle. This molecular analysis of rAAV persistence in NHP provides a promising basis for a reliable genotoxic risk assessment of rAAV in clinical trials.
authors.affiliation: National Center for Tumor Diseases Heidelberg, Department of Translational Oncology, German Cancer Research Center, Heidelberg, Germany.
authors.forename: Ali
authors.initials: A
authors.lastname: Nowrouzi
authors.forename: Magalie
authors.initials: M
authors.lastname: Penaud-Budloo
authors.forename: Christine
authors.initials: C
authors.lastname: Kaeppel
authors.forename: Uwe
authors.initials: U
authors.lastname: Appelt
authors.forename: Caroline
authors.initials: C
authors.lastname: Le Guiner
authors.forename: Philippe
authors.initials: P
authors.lastname: Moullier
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
authors.forename: Richard O
authors.initials: RO
authors.lastname: Snyder
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D000229:Dependovirus / Q000235:genetics*; D018628:Gene Dosage; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008099:Liver; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000821:virology; D011323:Primates / Q000378:metabolism* / Q000821:virology; D011533:Proviruses / Q000235:genetics; D011995:Recombination, Genetic; D016662:Virus Integration