title: Ribosomal DNA integrating rAAV-rDNA vectors allow for stable transgene expression.
abstract: Although recombinant adeno-associated virus (rAAV) vectors are proving to be efficacious in clinical trials, the episomal character of the delivered transgene restricts their effectiveness to use in quiescent tissues, and may not provide lifelong expression. In contrast, integrating vectors enhance the risk of insertional mutagenesis. In an attempt to overcome both of these limitations, we created new rAAV-rDNA vectors, with an expression cassette flanked by ribosomal DNA (rDNA) sequences capable of homologous recombination into genomic rDNA. We show that after in vivo delivery the rAAV-rDNA vectors integrated into the genomic rDNA locus 8-13 times more frequently than control vectors, providing an estimate that 23-39% of the integrations were specific to the rDNA locus. Moreover, a rAAV-rDNA vector containing a human factor IX (hFIX) expression cassette resulted in sustained therapeutic levels of serum hFIX even after repeated manipulations to induce liver regeneration. Because of the relative safety of integration in the rDNA locus, these vectors expand the usage of rAAV for therapeutics requiring long-term gene transfer into dividing cells.
authors.affiliation: Stanford University, Departments of Pediatrics and Genetics, Stanford, California 94305-5164, USA.
authors.forename: Leszek
authors.initials: L
authors.lastname: Lisowski
authors.forename: Ashley
authors.initials: A
authors.lastname: Lau
authors.forename: Zhongya
authors.initials: Z
authors.lastname: Wang
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.forename: Feijie
authors.initials: F
authors.lastname: Zhang
authors.forename: Markus
authors.initials: M
authors.lastname: Grompe
authors.forename: Mark A
authors.initials: MA
authors.lastname: Kay
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004275:DNA, Ribosomal / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D005164:Factor IX / Q000235:genetics / Q000378:metabolism; D005260:Female; D018628:Gene Dosage; D015870:Gene Expression; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D041681:NIH 3T3 Cells; D010957:Plasmids; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D014161:Transduction, Genetic; D019076:Transgenes; D016662:Virus Integration
chemical_list: D004275:DNA, Ribosomal; D005164:Factor IX