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David Dickey

Mentor: Joseph Zabner, M.D.
Lab Room: 440 EMRB
Lab Phone: 319-335-8457

Generation of Novel AAV Vectors for Cystic Fibrosis Gene Therapy.

Adeno-associated viruses (AAV) are promising vectors for use in gene therapy due to their high safety profile in human patients, and their ability to infect multiple cell types efficiently resulting in long term transgene expression. We are interested in using AAV for gene therapy in the lungs of patients with cystic fibrosis (CF), however, the AAV serotypes found in nature do not efficiently transduce human airway. Changes to the viral capsid may be sufficient to improve the targeting and transduction of airway. However, due to the complexity of each step, insufficient information is available for significant improvements through rational design. Our lab has employed a novel evolutionary strategy to increase the diversity of the AAV capsid and then select them on a polarized human airway model system. Briefly, viral DNA shuffling and error-prone PCR created a library containing ~106 unique chimeric AAV capsid sequences. This viral library was used to infect HAE from the apical side followed by infection with the helper wild-type adenovirus on the basolateral side to allow AAV replication. After five rounds of increasingly stringent selective pressures and two rounds of mutagenesis, recovery of the evolved AAV progeny was ~550-fold higher than the parental virus AAV2. Surprisingly, a single solution emerged. AAV2.5T is a chimera of AAV2 and AAV5, with amino acids 1-128 from AAV2 and amino acids 129-725 from AAV5 with a single point mutation (A581T). Transduction of HAE with recombinant AAV2.5T encoding the luciferase reporter gene resulted in expression that was 100-fold higher than AAV2-Luc, 10-fold higher than AAV5-Luc, and 20-fold higher than AAV9-Luc. This virus was able to correct the CF defect in airway epithelia isolated from patients with CF. My project is currently focused on determining the mechanism of how AAV2.5T is able to transduce HAE more effectively than parental AAV2 and AAV5. I will determine the receptor for AAV2.5T and whether it has improved intracellular trafficking compared to AAV2 and AAV5. I will also focus on making additional alterations to AAV2.5T which may result in even better transduction of HAE. For example, it has been found that point mutations in surface exposed tyrosines in the capsid of AAV2 lead to high-efficiency transduction at lower doses in vitro and in vivo. This was due to avoiding phosphorylation of the tyrosines and subsequent ubiquitination and proteasomal degradation of the capsid. Site-directed mutagenesis will be used to alter the surface exposed tyrosines in AAV2.5T and gene transfer will be evaluated in human airway epithelial cultures. Ongoing evolution with more complicated capsid libraries is also in progress and is expected to yield additional candidate AAV’s for gene therapy. My results so far show that directed evolution coupled with the proper clinically relevant and human-specific selection platform can effectively generate a highly efficient AAV vector capable of correcting the CF C1- transport defect to levels comparable to non-CF epithelia even at very low doses. Moreover, this general approach enables the development of ‘designer’ gene delivery vectors under clinically desirable selective pressures that will continue to yield exciting new candidates for human gene therapy.


Dickey DD, Excoffon KJ, Young KR, Parekh KR, Zabner J. Hoechst increases adeno-associated virus-mediated transgene expression in airway epithelia by inducing the cytomegalovirus promoter. J Gene Med. 2012 Jun;14(6):366-73. doi:10.1002/jgm.2632. PubMed PMID: 22610695.

Dickey DD, Excoffon KJ, Koerber JT, Bergen J, Steines B, Klesney-Tait J, Schaffer DV, Zabner J. Enhanced sialic acid-dependent endocytosis explains the increased efficiency of infection of airway epithelia by a novel adeno-associated virus. J Virol. 2011 Sep;85(17):9023-30. Epub 2011 Jun 22. PubMed PMID: 21697483; PubMed Central PMCID: PMC3165813.

Excoffon KJ, Koerber JT, Dickey DD, Murtha M, Keshavjee S, Kaspar BK, Zabner J, Schaffer DV. Directed evolution of adeno-associated virus to an infectious respiratory virus. Proc Natl Acad Sci U S A. 2009 Mar 10;106(10):3865-70. Epub 2009 Feb 23. PubMed PMID: 19237554; PubMed Central PMCID: PMC2646629.