Surface Loop Dynamics of a Parvovirus Public Deposited

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Last Modified
  • March 22, 2019
Creator
  • DiPrimio, Nina
    • Affiliation: School of Medicine, Department of Pharmacology
Abstract
  • In order to understand Adeno-associated virus and its potential as a gene delivery vehicle, we are obligated to characterize virus structure-function relationships and their ability to be manipulated for efficient gene delivery. In this dissertation, multiple capsid surface loops were characterized in order to understand their roles in the virus life cycle and the potential for advancement of viral gene therapy. Primarily, the HI loop, surrounding the five-fold pore on the capsid surface was characterized. Via domain swapping between serotypes, mutagenesis and biochemical analyses, it was determined that the HI loop is crucial for VP assembly. Specifically, a phenylalanine-proline interaction between subunits is necessary for maintaining full length VP1 incorporation into the intact particle during capsid assembly. From these studies, it was determined that the HI loop is highly plastic and certain amino acids are amenable to alteration. Therefore, the HI loop was substituted with alternative motifs including hexa-histidine. Not only did we generate vectors capable of metal ion affinity purification, but also conjugation to Ni-NTA nanogold beads for EM applications. Additionally, the hexa-histidine viruses display unique profiles in vivo, with tissue detargeting capabilities, modulated by altering the number of hexa-histidine tags on the surface. This alteration in targeting may be due to capsid stability but the exact mechanism remains to be determined, however, this finding may add to the development of liver detargeting vectors for the treatment of specific genetic disorders. Post characterization of the five-fold surface loop, interests expanded to amino acid position 265 within surface loop I between the five-fold and two-fold axes, deemed important in an alternative stage of the virus life cycle, infectivity. Upon AAV2 and AAV1 265 insertion and substitution, vectors display remarkable enhancements in liver and muscle transduction. Additionally, amino acid 265 substitution rescues infectivity of AAV2i8, a vector incapable of transducing the liver. Based on further characterization, mutation of position 265 enhances the ability of viral particles to enter cells, gaining tropism in multiple tissue types for potential use as gene therapy vectors. Therefore, the aforementioned data transcend virus biology to vectorology.
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  • In Copyright
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  • ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology
Advisor
  • Samulski, R. Jude
Degree granting institution
  • University of North Carolina at Chapel Hill
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