Journal article
Consequences of DNA-dependent protein kinase catalytic subunit deficiency on recombinant adeno-associated virus genome circularization and heterodimerization in muscle tissue
Journal of virology, Vol.77(8), pp.4751-4759
04/2003
DOI: 10.1128/JVI.77.8.4751-4759.2003
PMCID: PMC152118
PMID: 12663782
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.
Details
- Title: Subtitle
- Consequences of DNA-dependent protein kinase catalytic subunit deficiency on recombinant adeno-associated virus genome circularization and heterodimerization in muscle tissue
- Creators
- Dongsheng Duan - University of MissouriYongping Yue - Department of Molecular Microbiology and ImmunologyJohn F Engelhardt - University of Iowa
- Resource Type
- Journal article
- Publication Details
- Journal of virology, Vol.77(8), pp.4751-4759
- DOI
- 10.1128/JVI.77.8.4751-4759.2003
- PMID
- 12663782
- PMCID
- PMC152118
- ISSN
- 0022-538X
- eISSN
- 1098-5514
- Grant note
- HL58340 / NHLBI NIH HHS R01 HL058340 / NHLBI NIH HHS
- Language
- English
- Date published
- 04/2003
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering; Anatomy and Cell Biology; Radiation Oncology; Internal Medicine
- Record Identifier
- 9984288580502771
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