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Journal of Virology, April 2003, p. 4751-4759, Vol. 77, No. 8
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.8.4751-4759.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Consequences of DNA-Dependent Protein Kinase Catalytic Subunit Deficiency on Recombinant Adeno-Associated Virus Genome Circularization and Heterodimerization in Muscle Tissue

Dongsheng Duan,1,2* Yongping Yue,1 and John F. Engelhardt3,4

Department of Molecular Microbiology and Immunology,1 Program in Molecular Biology, School of Medicine, The University of Missouri, Columbia, Missouri,2 Department of Anatomy and Cell Biology,3 Department of Internal Medicine and Gene Therapy Center, School of Medicine, The University of Iowa, Iowa City, Iowa4

Received 10 September 2002/ Accepted 10 January 2003

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 ~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 ß-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.

* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, The University of Missouri, School of Medicine, 1 Hospital Dr., Room M610G, MSB, Columbia, MO 65212. Phone: (573) 884-9584. Fax: (573) 882-4287. E-mail: duand{at}health.missouri.edu.

Journal of Virology, April 2003, p. 4751-4759, Vol. 77, No. 8
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.8.4751-4759.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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