title: AAV recombineering with single strand oligonucleotides.
abstract: Adeno-associated virus (AAV) transduction initiates a signaling cascade that culminates in a transient DNA damage response. During this time, host DNA repair proteins convert the linear single-strand AAV genomes to double-strand circular monomers and concatemers in processes stimulated by the AAV inverted terminal repeats (ITRs). As the orientation of AAV genome concatemerization appears unbiased, the likelihood of concatemerization in a desired orientation is low (less than 1 in 6). Using a novel recombineering method, Oligo-Assisted AAV Genome Recombination (OAGR), this work demonstrates the ability to direct concatemerization specifically to a desired orientation in human cells. This was achieved by a single-strand DNA oligonucleotide (oligo) displaying homology to distinct AAV genomes capable of forming an intermolecular bridge for recombination. This DNA repair process results in concatemers with genomic junctions corresponding to the sequence of oligo homology. Furthermore, OAGR was restricted to single-strand, not duplexed, AAV genomes suggestive of replication-dependent recombination. Consistent with this process, OAGR demonstrated oligo polarity biases in all tested configurations except when a portion of the oligo targeted the ITR. This approach, in addition to being useful for the elucidation of intermolecular homologous recombination, may find eventual relevance for AAV mediated large gene therapy.
authors.affiliation: UNC Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Hirsch
authors.forename: Francesca
authors.initials: F
authors.lastname: Storici
authors.forename: Chengwen
authors.initials: C
authors.lastname: Li
authors.forename: Vivian W
authors.initials: VW
authors.lastname: Choi
authors.forename: R Jude
authors.initials: RJ
authors.lastname: Samulski
mesh_terms: D002460:Cell Line; D004249:DNA Damage; D004261:DNA Replication; D004279:DNA, Viral; D000229:Dependovirus / Q000235:genetics*; D005821:Genetic Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D009841:Oligonucleotides / Q000235:genetics*; D011995:Recombination, Genetic; D015398:Signal Transduction
chemical_list: D004279:DNA, Viral; D009841:Oligonucleotides; D049452:Green Fluorescent Proteins