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Endell, Jan (2006): Generation of enhanced gene delivery vectors by directed evolution of adeno-associated virus. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Despite promising advance in the development of viral vectors based on AAV for human gene therapy, several major hurdles for a more general use remain. Among these, efficient in vivo applications are limited by the high prevalence of neutralizing antibodies in the human population, which can reduce or eliminate transgene expression. A successful prevention of antibody-mediated vector neutralisation requires the modification of specific epitopes of the viral capsid responsible for Ab binding. The aim of this work was to demonstrate that immune-escaping capsid variants can be generated through genetic modifications of the virus by taking advantage of combinatorial engineering and directed evolution protocols. A library of 107 AAV mutants carrying random point mutations scattered throughout the capsid gene of AAV was created by error prone PCR and screened for clones that were able to avoid neutralization by AAV-neutralizing human sera. Three mutants carrying the mutations R459G, R459K and N551D respectively and a double mutant with a combined R459K/N551D mutation were strongly enriched after the selection procedure. Characterisation of these clones showed an immune-escaping phenotype for all mutants. However, the combination mutant proved to be superior in both evasion of neutralization and infectivity, leading to the assumption that multiple mutations convey enhanced effects. Therefore, the remaining pool was subjected to DNA shuffling and additional error prone PCR, yielding a second-generation library, which was screened for further improved phenotypes. In this context, a method which we called evolution monitoring was devised allowing optimization of several experimental conditions that are typically critical for successful outcome of library panning. These refinements yielded novel variants with further enhanced immune-escape abilities and infectivity in comparison to previously selected mutants. Finally, obtained data suggests an enormous potential for using the here developed tools to study infection biology of viruses by reverse genetics. This work showed for the first time that error prone PCR and DNA shuffling can be successfully applied for genetic engineering of a virus by a directed evolution approach. In principle, using appropriate selection protocols these techniques should be adaptable for addressing a wide variety of challenges concerning AAV in particular and virology in general.