Combinatorial selection of efficient and cell-type specific adeno-associated virus vectors (AAV) for human gene therapy
Final Report Abstract
The success of gene therapy applications using viral vectors relies on the availability of efficient and selective gene transfer systems that deliver therapeutic genetic information in target cells efficiently and avoiding transduction of neighbouring tissues at the same time. Currently available viral vectors often lack selectivity and can infect a wide range of cell types and tissues. Therefore, we are developing novel evolutionary techniques to select modified viruses based on adenoassociated virus of type 2 (AAV2) that infect target cells by specific and selective virus-receptor interactions. At the same time, we are establishing selection protocols to identify AAV variants that escape neutralization by pre-existing antibodies that can be used to treat patients that have been infected by AAV natural serotypes in the past. During the here described experiments we validated an in vitro method for the isolation of AAV variants with augmented selectivity for specific target cells. The mutants identified in this study can infect in vitro melanoma cells with up to 4-fold increased selectivity in comparison to wt AAV. Such vectors hold the potential to increase efficiency and safety of gene transfer to malignant cells in vivo. The negative selection protocol established in the funded period can be applied to select target selective vectors with tropism redirected against different cell types. The anticipated goal of engineering AAV vectors for the transduction of B-CLL cells has not been achieved yet. Transduction of these cells appears to be limited by other factors than weak receptor recognition, and experiments conducted to investigate these factors indicated that antibody neutralization is likely to be a major impediment. Therefore, we established a new protocol for the generation and screening of AAV mutants that escape neutralization by humoral immunity. These mutants remain infectious in the presence of serum concentrations up to 10-15%. The benefits of using these vectors in vivo in pre-immunized animals and in vitro for the transduction of B-CLL cells are currently being assessed in comparison to wt AAV-2 vectors. Finally, our findings demonstrate the importance of optimizing amino acid identity at each mutated site in order to control capsid phenotype of engineered vectors. This suggests that the next efforts to generate vectors with specific tropism should focus on employing viral libraries with randomized regions that are restricted to short tropism-relevant regions in order to increase the chance to represent "good" clones in the initial library.
Publications
- Optimization of stealth adeno-associated virus vectors by randomization of immunogenic epitopes. Virology 2009
S. Maersch, A. Huber, H. Büning, M. Hallek and L. Perabo
