title: Hybrid adeno-associated virus bearing nonhomologous inverted terminal repeats enhances dual-vector reconstruction of minigenes in vivo.
abstract: We have previously demonstrated that hybrid adeno-associated viral (AAV) vectors bearing nonhomologous inverted terminal repeats (ITRs) enhance directional intermolecular recombination and the efficiency of dual-AAV vector trans-splicing in cultured cells. Using hybrid-ITR vectors carrying two exons of a lacZ minigene, we demonstrate that this dual-vector approach also mediates higher levels (3- to 6-fold) of gene reconstitution in mouse skeletal muscle, liver, and heart. Inhibition of the proteasome by systemic administration of Doxil (Food and Drug Administration-approved lipid-formulated doxorubicin) further enhanced dual-vector trans-splicing 6- to 12-fold in two mouse strains. Hence, using hybrid-ITR AAV vectors in combination with proteasome modulation enhanced dual-vector delivery of a transgene approximately 36-fold over the current dual-vector trans-splicing approaches. These data provide in vivo evidence that ITR sequence-dependent homologous recombination, rather than nonhomologous end joining, is the predominant mechanism for AAV genome heterodimerization. Hence, enhanced directional recombination provided by hybrid-ITR vectors may be a useful in vivo strategy for improving dual-vector delivery of transgenes larger than the AAV packaging limit.
authors.affiliation: Department of Anatomy and Cell Biology, University of Iowa School of Medicine, Iowa City, IA 52242, USA.
authors.forename: Ziying
authors.initials: Z
authors.lastname: Yan
authors.forename: Diana C M
authors.initials: DC
authors.lastname: Lei-Butters
authors.forename: Yulong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Roman
authors.initials: R
authors.lastname: Zak
authors.forename: John F
authors.initials: JF
authors.lastname: Engelhardt
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D002460:Cell Line; D000229:Dependovirus; D004317:Doxorubicin / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral / Q000187:drug effects / Q000235:genetics; D005822:Genetic Vectors; D016679:Genome, Viral / Q000235:genetics; D007763:Lac Operon; D051379:Mice; D020079:Terminal Repeat Sequences; D020040:Trans-Splicing / Q000187:drug effects; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D000903:Antibiotics, Antineoplastic; D004317:Doxorubicin