title: Consequences of DNA-dependent protein kinase catalytic subunit deficiency on recombinant adeno-associated virus genome circularization and heterodimerization in muscle tissue.
abstract: Circular concatemerization of the recombinant adeno-associated virus (rAAV) genome has been suggested as the predominant process facilitating long-term rAAV transduction in muscle. A recent study (S. Song, P. J. Laipis, K. I. Berns, and T. R. Flotte, Proc. Natl. Acad. Sci. USA 98:4084-4088, 2001) with SCID mice, which are defective in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), has suggested that DNA-PKcs regulates the removal of free rAAV vector ends in muscle tissue. In the present study, we have sought to evaluate whether a lack of DNA-PKcs activity reduces circularization of rAAV genomes in SCID muscle and whether such a reduction alters the directivity of heterodimerization. Consistent with the previous report, linear rAAV genomes and free vector ends were detected only in DNA-PKcs-deficient muscle by Southern blotting. Appreciable amounts of circular rAAV genomes were detected in both DNA-PKcs-deficient and wild-type muscle samples by Southern blotting and bacterial trapping experiments. The existence of double-D inverted terminal repeat circular intermediates in SCID and wild-type muscles was also supported by their sensitivity to T7 endonuclease I digestion. However, DNA-PKcs-deficient muscle did demonstrate a approximately 50% reduction in the abundance of rescued circular genomes, despite equivalent levels of single rAAV transduction seen in wild-type animals. Dual trans-splicing lacZ vectors were used to functionally evaluate directional head-to-tail intermolecular viral genome concatamerization in vivo. Although AAV genomes are processed differently in SCID and wild-type muscles, a comparable level of trans-splicing-mediated beta-galactosidase expression was observed in both strains, suggesting that both circular and linear AAV concatemers may have contributed to the trans-splicing-mediated transgene expression. In summary, we have shown that SCID skeletal muscle retains a fairly high capacity to form circular genomes, despite a significant increase in linear vector genomes. Furthermore, the alteration in equilibrium between circular and linear concatemer genomes caused by the lack of DNA-PKcs activity does not appear to significantly affect the efficiency of dual-vector gene expression from head-to-tail linear and/or circular heterodimers.
authors.affiliation: Department of Molecular Microbiology and Immunology and Program in Molecular Biology, School of Medicine, The University of Missouri, Columbia, Missouri 65212, USA. duand@health.missouri.edu
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Duan
authors.forename: Yongping
authors.initials: Y
authors.lastname: Yue
authors.forename: John F
authors.initials: JF
authors.lastname: Engelhardt
mesh_terms: D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D004270:DNA, Circular / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D000229:Dependovirus / Q000201:enzymology / Q000235:genetics*; D019281:Dimerization; D016679:Genome, Viral; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000821:virology; D009690:Nucleic Acid Conformation; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014161:Transduction, Genetic
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases