aav recombineering with single strand oligonucleotidesaav recombineering与单链寡核苷酸.pdfVIP

AAV Recombineering with Single Strand Oligonucleotides 1 3 1 4 1,2 Matthew L. Hirsch , Francesca Storici , Chengwen Li , Vivian W. Choi , R. Jude Samulski * 1 UNC Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 2 Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 3 School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America, 4 Harvard Medical School, Cambridge, Massachusetts, United States of America 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-depend