Pharmacological and Biophysical Dissections of the Trafficking and Infection of Adeno-Associated Virus (AAV) In Vitro and In Vivo
Public Deposited- Last Modified
- March 22, 2019
- Creator
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Xiao, Pingjie
- Affiliation: School of Medicine, Department of Cell Biology and Physiology
- Abstract
- Adeno-associated virus (AAV) is a defective and non-pathogenic human parvovirus that is dependent on the co-infection of a helper virus, such as Adenovirus or Herpes Virus, to fulfill its productive life cycle. While many traits of this virus have made it an attractive vector for gene therapy as well as the wide application in over 100 clinical trials, there remains a great need to further improve the performance of AAV in gene delivery. As a most promising solution to fit such need, development of novel AAV vectors with improved efficiency on cellular trafficking and processing was partially achieved by several strategies in the last decade, including isolation of natural serotypes, directed evolution, and rational design. Being a most effective approach for the development of new AAV vectors in the future, rational design requires extensive knowledge on AAV-host interaction, which is mainly a multi-step trafficking and intracellular processing from cell surface binding to nuclear entry. However, much is unknown about the details of interactions between AAV and its host, which has limited one's ability to improve the delivery efficacy of AAV vectors and therefore restricted their medical applications. The primary goal of this dissertation is to advance our current knowledge of AAV-host interactions by developing new methodologies and providing more insights on how virion components and host machineries at molecular level affect the infectious pathway of AAV. Typically, a successful transduction is achieved upon the accomplishment of virions on cell surface attachment, endocytosis and endosomal sorting, cytoplasmic trafficking, nuclear targeting, and genome processing. Prior to this work, an efficient method to quantitatively evaluate the intracellular trafficking of AAV particles is not available. Additionally, it was unknown how the AAV traverses the crowded cytoplasm in host cells prior to the nuclear entry. While microtubules (MTs) have been reported to facilitate the trafficking of many other viruses, it was unclear whether and how this cellular machinery may regulate the intracellular behavior of AAV. Therefore, we set out to build on the known foundation of AAV biology and hypothesize that MTs play an important role on AAV infection especially the cytoplasmic trafficking of this virus. In this thesis, we have established a method of computer-assisted quantitative 3D bio-distribution microscopy that samples the whole population of fluorescently-labeled vectors and documents their trafficking routes through cells and tissues. This method shall facilitate a quantitative evaluation of the effects of pharmacological reagents and vector variants on the delivery performance of viral vectors. With this method, we have demonstrated that AAV particles exploit MT-mediated endosome transportation to traverse the dense cytoplasm to reach the host nucleus. In addition, we have discovered a new cellular barrier composed of MTs at MTOC region that limits the nuclear entry and transduction of AAV virions, defining a novel defense mechanism by which host cells restrain viral invasion. On the basis of above findings, we have proposed a model to better illustrate the infectious pathway of AAV and its fine-tuning by MTs. Moreover, we have also documented the trafficking of AAV in animal tissues, providing new insights to the AAV biology and vectorology in vivo. While focusing on how AAV traffics through the subcellular maze, this work intersects areas of cell biology, biophysics, virology, and gene delivery. This work provides valuable research tools, rationales, and assays for future exploration of the details of AAV trafficking and will contribute to the knowledge of AAV biology, facilitating the rational development of gene therapy vectors.
- Date of publication
- May 2013
- DOI
- Resource type
- Rights statement
- In Copyright
- Advisor
- Samulski, R. Jude
- Degree
- Doctor of Philosophy
- Graduation year
- 2013
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