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Diverse Effects of DNA Repair Pathways on the Outcome of Recombinant Adeno-Associated Virus (rAAV) Vector Gene Delivery

Cataldi, Marcela Patricia
http://rave.ohiolink.edu/etdc/view?acc_num=osu1303842573

Abstract Details

2011, Doctor of Philosophy, Ohio State University, Molecular, Cellular and Developmental Biology.
Because of Adeno-Associated Virus (AAV) nonpathogenicity and ability to promote sustained, long-term transgene expression in a wide variety of tissues including the brain, liver, muscle, retina, and vasculature, several recombinant AAV (rAAV) serotypes are emerging as attractive vectors for gene therapy. The linear DNA genomes of AAV are acted upon by multiple DNA repair and recombination pathways once released into the host nucleus. The palindromic terminal repeats (TR) on both ends of the AAV genome, which form DNA hairpin (HP) structures, serve as substrates for intramolecular and intermolecular recombination, leading to circularization and concatemer formation, respectively. Infrequently, AAV TRs can also recombine with the host genome, leading to chromosomal integration. However, the mechanism of recombination between AAV TRs is still unclear, and with respect to integration, recombination raises concern for genotoxicity. I have compared the fates of single-stranded rAAV (ssAAV) and self-complementary (scAAV) genomes in cell lines deficient in each of three signaling factors, ATM, ATR, and DNA-PKcs, which orchestrate major DNA double-strand break (DSB) repair pathways. The results suggest that there are qualitative and quantitative differences between both vectors in terms of their interactions with different host DNA repair and recombination pathways. A striking and unexpected observation from these experiments was that in cells deficient in ATM, transduction scored by GFP expression was increased relative to wild-type (WT) cells for both rAAV vectors. The augmented transduction was not reflected in Southern blots of nuclear vector DNA, suggesting that interactions with ATM lead to silencing in normal cells. The additional functional genomes in ATM-/- cells remained linear, and the number of circularized genomes was not affected by the mutation, consistent with compartmentalization of genomes into different DNA repair pathways. To investigate whether the TR HP has any specific effect on recombination, I compared DNA substrates containing the TR sequences in an open DNA duplex conformation or in a covalently closed hairpin conformation, or containing blunt DNA ends with no TR sequence. These DNA substrates were transfected into either WT or DNA-repair deficient cells. DNA molecules with the TR sequences constrained in the hairpin conformation at one or both ends were subject to a loss of gene expression, which was partially relieved in ATM-/- cells. This ATM-mediated effect was not due to a loss of DNA, suggesting that a single AAV TR in the hairpin conformation in cis is necessary and sufficient to mediate the ATM-dependent silencing of gene expression from rAAV genomes. The results also indicate that DNA substrates with AAV TR in a hairpin conformation circularize and integrate with the same efficiency as other forms of DNA ends in WT cells. Therefore, the specificity of the recognition of the HP structure of rAAV vector genomes by DNA repair/recombination pathways leads to loss of functional genomes rather than an enhancement of recombination. I further investigated rAAV recombination by comparing circularization and integration in dividing and non-dividing cultured cells. The results indicated that neither circularization nor integration requires active cell division, and that integration occurs rapidly after infection, over the same time period as circularization.
Douglas McCarty, PhD (Advisor)
Samir Acharya, PhD (Committee Member)
Reed Clark, PhD (Committee Member)
Deborah Parris, PhD (Committee Member)
154 p.
Molecular Biology; Virology
adeno-associated virus; gene therapy; DNA recombination; rAAV vector; DNA viral vectors

Recommended Citations

Citations

  • Cataldi, M. P. (2011). Diverse Effects of DNA Repair Pathways on the Outcome of Recombinant Adeno-Associated Virus (rAAV) Vector Gene Delivery [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1303842573

    APA Style (7th edition)

  • Cataldi, Marcela. Diverse Effects of DNA Repair Pathways on the Outcome of Recombinant Adeno-Associated Virus (rAAV) Vector Gene Delivery. 2011. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1303842573.

    MLA Style (8th edition)

  • Cataldi, Marcela. "Diverse Effects of DNA Repair Pathways on the Outcome of Recombinant Adeno-Associated Virus (rAAV) Vector Gene Delivery." Doctoral dissertation, Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1303842573

    Chicago Manual of Style (17th edition)

osu1303842573
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© 2011, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.