Dissertation
Elucidating the factors determining efficient transduction of human airway epithelia by AAV gene therapy vectors
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2020
DOI: 10.25820/etd.007195
Abstract
Vectors derived from adeno-associated virus (AAV) are FDA approved to transfer genes to the retina for treatment of Leber’s congenital amaurosis. AAVs have also been proposed to carry genetic therapy for cystic fibrosis (CF). AAV2 was safe when aerosolized into the lungs of people with CF in clinical trials, but no evidence of transgene expression was detected. AAV2 transduction is also inefficient on primary human airway epithelia (HAE) models, possibly due to a lack of appropriate binding partners on the apical (luminal) side. We previously used directed evolution to select AAV2.5T, a chimera of AAV2 and AAV5 capsids. Transduction by AAV2.5T is significantly more efficient than AAV2 or AAV5 on apical HAE. Further, unlike AAV2 or AAV5, AAV2.5T binds specifically to abundant apical partners on HAE. Recently, cellular KIAA0319L was determined essential for transduction by most naturally occurring AAVs, and renamed AAVR. We hypothesized AAVR was not apically expressed, and that AAV2.5T evolved to use a non-AAVR receptor on apical HAE. AAVR is expressed in HAE, but never detected in the apical membrane. Overexpressed AAVR is detected in the basolateral membrane, and increases basolateral transduction preferentially. Anti-AAVR antibodies do not reduce AAV2.5T transduction of apical HAE, but moderately reduce basolateral transduction. AAVR antibodies virtually eliminate transduction by AAV2. These data suggest AAVR is localized to the basolateral membrane in HAE and explain AAV2’s clinical trial inefficiency. Further, on HAE, AAV2.5T uses basolateral AAVR and an alternative receptor, which AAV2.5T exploits for efficient apical transduction.
In HEK cells, AAV2 transduction was more efficient than AAV2.5T and AAV5, while in HeLa cells AAV2.5T was most efficient. In both cell lines, antibodies blocked infection by all vectors in a dose dependent manner. However, AAV2.5T was less susceptible to block by high dose antibodies on HeLa. CRISPR knockout of AAVR produced HEK and HeLa cells refractory to infection by AAV2 and AAV5. AAV2.5T was unable to infect HEK cells lacking AAVR, but could infect AAVR knockout HeLa. These findings demonstrated the presence of a non-AAVR receptor facilitating AAV2.5T infection in HeLa cells, but not in HEK cells. We hypothesized the same non-AAVR receptor would be found in cell lines and HAE. A cell line screen, followed by bioinformatic analyses, led us to hypothesize the non-AAVR receptor was exploited by other respiratory viruses. We then interrogated measles virus receptors and found SLAMF1 confers AAV2.5T apical entry in polarized Vero cells. Interestingly, SLAMF1 is not expressed in HAE. Our preliminary data suggest a related protein, SLAMF7, may facilitate the efficient apical transduction of HAE by AAV2.5T.
These findings have important implications. SLAMF7 may provide a target for personalized gene therapy in CF airways. The structural alterations to the AAV2.5T capsid could inform vector designs targeting other cell types. Lastly, SLAMF7 should be investigated as a potential entry receptor for measles virus.
Details
- Title: Subtitle
- Elucidating the factors determining efficient transduction of human airway epithelia by AAV gene therapy vectors
- Creators
- Bradley A. Hamilton
- Contributors
- Joseph Zabner (Advisor)Mahmoud Abou Alaiwa (Committee Member)Eric Taylor (Committee Member)Christie Thomas (Committee Member)Charles Yeaman (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Molecular and Cellular Biology
- Date degree season
- Spring 2020
- DOI
- 10.25820/etd.007195
- Publisher
- University of Iowa
- Number of pages
- xi, 147 pages
- Copyright
- Copyright 2020 Bradley A. Hamilton
- Language
- English
- Date submitted
- 05/08/2020
- Date approved
- 06/30/2023
- Description illustrations
- Illustrations, tables, graphs, charts
- Description bibliographic
- Includes bibliographical references (pages 132-147).
- Academic Unit
- Interdisciplinary Graduate Program in Molecular Medicine
- Record Identifier
- 9984425393902771
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