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Recombinant Adeno-associated Viral Vector Design Influences Genotoxic Potential

Westmoreland, Patrick Riley
http://orcid.org/0000-0003-1856-4181
http://rave.ohiolink.edu/etdc/view?acc_num=osu1514462220056427

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Molecular, Cellular and Developmental Biology.
Gene therapy utilizing recombinant adeno-associated virus (rAAV) has been shown to be a safe and effective treatment in mouse models of human disease. Albeit less likely to promote genotoxicity than obligate integrating vectors such as retroviruses, studies have shown an increase in hepatocellular carcinoma (HCC) in mice treated with rAAV. The focus of this study is to determine what features of rAAV vectors could contribute to tumor promotion by recovering vector genome junctions from HCC in male C3H/HeJ mice, in which liver tumors grow rapidly, to achieve the sensitivity needed to detect rAAV-associated oncogenic events. Several different rAAV vectors were designed to examine how the inclusion of introns, enhancer elements, promoter strength, and transgene expression influences tumorigenesis. Genomic DNA from tumors of mice treated with rAAV was enriched for vector sequence using a custom set of SureSelect RNA probes from Agilent. The enriched DNA was then sequenced on an Illumina HiSeq4000 sequencer. Vector integration sites were defined as any split read that aligned to both the vector genome and the mouse genome. Through this analysis 102 unique integration sites were identified from 130 tumors analyzed. 70 of the 102 unique integration sites were either in or near known proto-oncogenes. The overwhelming majority of those vector integration sites that were recovered came from mice treated with vectors derived from the Chicken ¿-actin(CBA) promoter. Interestingly, integration sites that were recovered from mice treated with scCMV-GFP vectors were all within the second intron of the proto-oncogene itch, and all had the GFP coding region deleted, thus containing only the CMV promoter and a portion of the SV40 intron. Furthermore, all but two integration sites that were recovered are hypothesized to cause the over-expression of their respective proto-oncogene. In only one instance was an intact expression cassette associated with a tumor which, in this case, caused cellular transformation by truncating the EGFR coding region, causing it to become constitutively active. Together, the data suggest that intact vectors pose little risk of activating proto-oncogenes. However, analysis of episomal vectors from normal liver tissue of mice treated with the scCMV-GFP vector showed 14% of vector genomes were less than full-length, and 2% of the vector sequence contained only the CMV promoter and the SV40 intron splice donor site, similar to the integrated structures recovered within itch. Additionally, half the mice also received a secondary treatment of a 43% partial hepatectomy. The hepatectomy removes any vector associated with the lobes that were removed and also causes synchronous cell division of hepatocytes as the liver regenerates. A decrease in the tumor incidence in hepatectomy-treated mice without was observed. This finding leads to the reassuring possibility that the rAAV treatment in young patients with actively growing livers cells or tissue does not pose a higher risk of oncogenesis as the liver continue to grow. Altogether, the data presented within this dissertation will allow for the development of rAAV vectors that pose the least risk to patients who receive them for various gene therapy applications.
Douglas McCarty (Advisor)
Patrick Green (Advisor)
Mark Peeples (Committee Member)
Scott Loiler (Committee Member)
143 p.
Biology; Molecular Biology
rAAV; Aden-associate virus; Viral Integration

Recommended Citations

Citations

  • Westmoreland, P. R. (2018). Recombinant Adeno-associated Viral Vector Design Influences Genotoxic Potential [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1514462220056427

    APA Style (7th edition)

  • Westmoreland, Patrick. Recombinant Adeno-associated Viral Vector Design Influences Genotoxic Potential. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1514462220056427.

    MLA Style (8th edition)

  • Westmoreland, Patrick. "Recombinant Adeno-associated Viral Vector Design Influences Genotoxic Potential." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1514462220056427

    Chicago Manual of Style (17th edition)

osu1514462220056427
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This open access ETD is published by The Ohio State University and OhioLINK.