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Optimization of design and production strategies for novel adeno-associated viral display peptide libraries

Gene Therapy volume 24, pages 470–481 (2017)Cite this article

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Abstract

Libraries displaying random peptides on the surface of adeno-associated virus (AAV) are powerful tools for the generation of target-specific gene therapy vectors. However, for unknown reasons the success rate of AAV library screenings is variable and the influence of the production procedure has not been thoroughly evaluated. During library screenings, the capsid variants with the most favorable tropism are enriched over several selection rounds on a target of choice and identified by subsequent sequencing of the encapsidated viral genomes encoding the library capsids with targeting peptide insertions. Thus, a high capsid-genome correlation is crucial to obtain the correct information about the selected capsid variants. Producing AAV libraries by a two-step protocol with pseudotyped library transfer shuttles has been proposed as one way to ensure such a correlation. Here we show that AAV2 libraries produced by such a protocol via transfer shuttles display an unexpected additional bias in the amino-acid composition which confers increased heparin affinity and thus similarity to wildtype AAV2 tropism. This bias may fundamentally impair the intended use of AAV libraries, discouraging the use of transfer shuttles for the production of AAV libraries in the future.

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Acknowledgements

We are grateful to Anna Oberle, Simon Schliffke and Nuray Akyüz, University Medical Center Hamburg-Eppendorf, Germany, for their technical support and expertise regarding the next-generation sequencing. We thank Hans O. Pinnschmidt, Institute of Medical Biometry and Epidemiology, University Medical Center Hamburg-Eppendorf for statistical consulting and we thank Jude Samulski, University of North Carolina, Chapel Hill, NC for kindly providing the plasmid pXX6 and This work was supported by the Deutsche Krebshilfe (grant 110902 to MT) and the Margarete Clemens Foundation (endowed professorship to MT).

Author contributions

Designed the experiments: JK, MT. Performed the experiments: JK, AH. Analyzed the data: JK, AH, MA. Discussed the data: JK, MA, MB, MT. Contributed material/technical equipment: TS, MB. Wrote the manuscript: JK, MT.

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Author notes

  1. M Trepel

    Present address: 4Current address: Augsburg Medical Center, Department of Hematology and Oncology, Augsburg, Germany.,

Authors and Affiliations

  1. Department of Oncology and Hematology, University Medical Center Hamburg-Eppendorf, Hubertus Wald Cancer Center, Hamburg, Germany

    J Körbelin, A Hunger, T Sieber, M Binder & M Trepel

  2. University Medical Center Hamburg-Eppendorf, Bioinformatics Core, Hamburg, Germany

    M Alawi

  3. Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology, Virus Genomics, Hamburg, Germany

    M Alawi

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  1. J Körbelin
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Correspondence to M Trepel.

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Körbelin, J., Hunger, A., Alawi, M. et al. Optimization of design and production strategies for novel adeno-associated viral display peptide libraries. Gene Ther 24, 470–481 (2017). https://doi.org/10.1038/gt.2017.51

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