Factors affecting the formation, stability, and expression of unintegrated lentiviral vector genomes Public Deposited

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Last Modified
  • March 21, 2019
Creator
  • Bayer, Matthew Alexander
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
Abstract
  • Lentiviral vectors present an attractive means of delivering therapeutic transgenes, as they deliver a relatively large, stably integrated and expressed genetic payload to both dividing and nondividing cells. However, with the genotoxic hazard of integrating vectors tragically illustrated by recent clinical trials using simple retroviral vectors, the necessity of developing a lentivector with minimal risk of insertional mutagenesis is clear. In fact, integrase-mutated lentivectors, which deliver only unintegrated, episomal vector genomes to target cells, have been in use for a number of years, and offer a means of stably expressing transgenes in nondividing cells. However, the means by which the various types of episomes (linear, 1-LTR circular, 2-LTR circular, and mutant circular) are formed, and the extent to which they are transcriptionally active, have not been thoroughly characterized in vitro or in vivo. This dissertation investigates the effect of cellular factors, such as DNA-repair proteins and cell-cycle status, vector factors, such as sequences in the vector's U3 region and polypurine tract (PPT), and organismal factors, such as target organ and in vivo stability, on the formation, stability, and expression of lentivector episomes. Interestingly, transduction of cell lines mutant for genes in the homologous-recombination pathway of DSB repair showed no change in 1-LTR circular-episome formation. Similarly, cells arrested in G1 phase of the cell cycle, which does not support HR, displayed unaffected levels of 1-LTR circles. Furthermore, vectors bearing a large deletion to the U3 region, which strongly increases episomal, though not integrated, transgene expression, saw no significant change in episome formation from that of a vector with a short U3 deletion. Conversely, vectors with a deleted PPT exhibited a dramatic increase in the relative abundance of 1-LTR circles, leading to reduced integrase-mediated and integrase-independent integration. Finally, examination of in vivo episome formation and expression indicated that the relative abundance of 1-LTR circular episomes increases over a three-week period in the liver, that episomes express trangenes stably for up to six months in the liver, and that episomes exhibit greater stringency of tissue-specific expression than integrated provirus.
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  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum in Genetics and Molecular Biology."
Advisor
  • Kafri, Tal
Degree granting institution
  • University of North Carolina at Chapel Hill
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  • Chapel Hill, NC
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  • Open access
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