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  • March 19, 2019
  • Albright, Blake
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
  • Adeno-associated virus (AAV) is currently the most widely used gene therapy vector for treating neurological diseases, showing promising results in preclinical and clinical studies. Nonetheless, many challenges limit effective gene transfer to the central nervous system (CNS) via the vasculature, which requires that AAV vectors cross the blood-brain barrier (BBB). As a consequence of sub-optimal transduction efficiency, current vectors must be administered at high dosages to achieve therapeutic CNS gene transfer and are limited by off-target tissue transduction/sequestration. Thus, developing vectors with improved efficiency and specificity is critical towards achieving widespread and therapeutic gene transfer to the CNS. This requires a better understanding of the structural determinants for AAV tropism and neurovascular transport – which is the primary aim for this dissertation. To achieve this, we generated a chimeric capsid library between two highly homologous serotypes – AAVrh.10, which crosses the BBB, and AAV1, which is limited to the vasculature. Through screening individual variants in vivo, computational analyses, and rational design, we mapped a footprint from the AAVrh.10 capsid which confers BBB transport and widespread CNS transduction when grafted onto AAV1. In this way, we engineered the novel and neurotropic AAV1RX capsid, which mediates robust gene transfer throughout the brain, with reduced glial and endothelial transduction, and is detargeted from peripheral tissues (i.e. liver). We hypothesized that the 1RX footprint may alter capsid-sialic acid (SIA) interactions in a way that promotes BBB traversal and CNS transduction. We tested this through functional characterization of several capsid variants with alterations in their SIA binding site. These capsids were functionally grouped according to their differential dependencies on cell-surface SIA for in vitro transduction and binding. Further evaluation in vivo revealed an inverse correlation between capsid-SIA interactions and BBB transport/CNS transduction. These experiments suggest that more moderate capsid-SIA interactions may facilitate, though only partially explain, the 1RX phenotype. Nonetheless, this led us to propose a Goldilocks model wherein the ideal glycan binding affinity, in conjunction with other contributing factors, impacts BBB traversal and CNS transduction. Thus, this dissertation sheds light on AAV determinants for BBB transport, providing a structure-guided platform for engineering improved CNS-targeted AAV vectors.
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  • In Copyright
  • Swanstrom, Ronald
  • Asokan, Aravind
  • Neher, Saskia
  • Song, Juan
  • Kafri, Tal
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2018

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