Abstract
Directed Evolution of AAV in Pig Airway Epithelia In Vivo
Molecular therapy, Vol.20(9), pp.24-24
09/01/2012
DOI: 10.1016/S1525-0016(16)37227-6
Abstract
Recently, a cystic fibrosis (CF) pig model was developed that demonstrates many of the hallmarks of the human disease, including lung disease. We therefore hypothesized that directed evolution of adeno-associated viruses (AAV) could be used to generate a novel AAV gene therapy vector for the airways of the CF pig. We used DNA shuffling and error-prone PCR of the cap genes of AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9, along with an AAV2 binding loop insertion library to create a library of approximately 10[and]7 unique AAV capsid variants. This virus library was pre-selected in vitro in pig airway epithelial cultures for three rounds, followed by mutagenesis for further diversification, and then subjected to three rounds of selection in vivo in the lungs of wild-type pigs. We recovered a single variant that was entirely composed of the AAV2 cap gene with five point mutations, which we called AAV2H22. We found that AAV2H22 transduced pig airway epithelial cultures in vitro approximately 240-fold better than AAV2. In contrast to AAV2, AAV2H22 bound the apical surface of pig airway epithelia with specificity, suggesting altered receptor binding, although, in cells lines, heparan sulfate proteoglycan is still be utilized as a receptor for the virus. AAV2H22 transduced pig alveolar epithelia much less efficiently than AAV2, and did not demonstrate increased transduction of human airway epithelia compared to AAV2, suggesting that AAV2H22 is optimized specifically for the transduction of cells in the airway epithelia of pigs.
Details
- Title: Subtitle
- Directed Evolution of AAV in Pig Airway Epithelia In Vivo
- Creators
- David DickeyBoulos NassarKatherine ExcoffonJames KoerberJamie BergenDavid SchafferJoseph Zabner
- Resource Type
- Abstract
- Publication Details
- Molecular therapy, Vol.20(9), pp.24-24
- DOI
- 10.1016/S1525-0016(16)37227-6
- ISSN
- 1525-0016
- eISSN
- 1525-0024
- Language
- English
- Date published
- 09/01/2012
- Academic Unit
- Pulmonary, Critical Care, and Occupational Medicine; Internal Medicine
- Record Identifier
- 9984363425802771
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