Abstract/Details

Characterization of adeno -associated virus 2 site -specific integration


2009 2009

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Abstract (summary)

Adeno-associated virus (AAV) is the only mammalian DNA virus known to be capable of establishing latency by integration into a specific site, called AAVS1, on Chromosome 19q13.4. The AAV cis and trans requirements for this process have been identified, yet, a complete picture has not emerged with regards to the precise mechanism. Specifically, the host protein requirements are not known, as well as how they could be interacting with the AAV genome.

This study has focused on two specific research objectives. The first objective was to determine if the AAV Rep protein, shown to be required for targeting site-specific integration, can minimize AAV random integration. The second objective was to study cellular repair proteins for an effect on AAV site specific integration.

My data has indicated that even in the presence of Rep, random integrations do take place, but, the limited number of identified AAV-cellular junctions precludes any definitive statements as to whether Rep can minimize random integration. In addition, I presented direct evidence that a protein called DNA-dependent protein kinase catalytic subunit (DNAPKcs) inhibits stable site-specific integration of single-stranded AAV Rep positive vectors. Moreover, the presence or absence of DNAPKcs did not affect the specific integration of self-complementary AAV Rep positive vectors, which also displayed more random integration. In addition, cellular repair proteins ligase I and ligase IV are not needed for AAV-AAVS1 junction formation, but the absence of ligase IV greatly reduced the frequency of AAV site-specific integration.

These findings contribute to a better understanding of the AAV site-specific integration process, suggesting that components of the non-homologous end joining pathway can modulate AAV site-specific integration. By systematically studying the different cellular repair proteins for an effect on specific integration, future work should be able to elucidate the mechanism. The outcome of such advancements would lead to an increased emphasis for the further development of AAV as a safe and efficient integrating vector for future gene therapy studies.

Indexing (details)


Subject
Genetics;
Microbiology;
Virology
Classification
0369: Genetics
0410: Microbiology
0720: Virology
Identifier / keyword
Biological sciences; Adeno-associated virus; Cellular factors; Gene therapy; Rep; Site-specific integration; Viral vectors
Title
Characterization of adeno -associated virus 2 site -specific integration
Author
Daya, Shyam
Number of pages
107
Publication year
2009
Degree date
2009
School code
0070
Source
DAI-B 70/07, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9781109274790
Advisor
Berns, Kenneth I.
University/institution
University of Florida
University location
United States -- Florida
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
3367415
ProQuest document ID
304879878
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/304879878
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