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Molecular Characterization of Adeno-Associated Virus in the Natural Host

Jensen, Ryan Lee
http://rave.ohiolink.edu/etdc/view?acc_num=osu1211229929

Abstract Details

2008, Doctor of Philosophy, Ohio State University, Integrated Biomedical Science.
Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. Based on the extensive in vitro data demonstrating site-specific integration of AAV, our hypothesis was that targeted integration would also occur in vivo. To that end, we tested 175 pediatric tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). Sequence analyses showed that 8 of the capsid sequences were AAV2-like (~98% amino acid identity), while the single spleen isolate was intermediate between serotypes 2 and 3. In these 9 samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay (LAM-PCR) to widen our search for integration events. Analysis of individual LAM-PCR products revealed that the AAV genomes were arranged predominately in a head-to-tail array, with deletions and extensive rearrangements in the inverted terminal repeat (ITR) sequences. A single AAV-cellular junction was identified from a tonsil sample and it mapped to a highly repetitive satellite DNA element on chromosome 1. Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extra-chromosomal forms. We used a novel amplification assay (linear rolling circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. We then go on to describe the molecular structure of several AAV molecular clones isolated from these pediatric tissues. DNA sequence analysis of the molecular clones revealed the ubiquitous presence of a double-D ITR structure, which suggests a mechanism by which the virus is able to maintain ITR sequence continuity and persist in the absence of host chromosome integration. To further define the molecular form of AAV persistence in a relevant animal model, we surveyed rhesus macaque tissues for the presence of wild-type AAV sequences and when detected, determined the molecular form of AAV persistence. To that end, we tested 28 monkey tissue samples from 9 animals and detected AAV sequence in 25 of the samples (89.3%). Significantly, in these 9 samples, we were unable to detect AAV integration in the macaque AAVS1 locus using a sensitive PCR assay designed to amplify viral-cellular DNA junctions. This was particularly notable given the high AAV copy number in several tissues. Given these data combined with our findings from human tissues, it seemed likely that the AAV genomes were persisting un-integrated and present predominately as extra-chromosomal forms. Finally, based on the knowledge that AAV persists as episomes, we go on to create novel AAV phenotypes with enhanced characteristics.
Virginia Sanders, PhD (Advisor)
Philip Johnson, MD (Committee Member)
Jeffrey Bartlett, PhD (Committee Member)
Douglas McCarty, PhD (Committee Member)
223 p.
Molecular Biology
Adeno-associated virus; AAV; Integration; Episomes

Recommended Citations

Citations

  • Jensen, R. L. (2008). Molecular Characterization of Adeno-Associated Virus in the Natural Host [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1211229929

    APA Style (7th edition)

  • Jensen, Ryan. Molecular Characterization of Adeno-Associated Virus in the Natural Host. 2008. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1211229929.

    MLA Style (8th edition)

  • Jensen, Ryan. "Molecular Characterization of Adeno-Associated Virus in the Natural Host." Doctoral dissertation, Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1211229929

    Chicago Manual of Style (17th edition)

osu1211229929
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© 2008, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.