Next Article in Journal
Circadian Gene Polymorphisms Associated with Breast Cancer Susceptibility
Previous Article in Journal
Provitamin A Biofortification of Durum Wheat through a TILLING Approach
Open AccessArticle

rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations

1
Cellular and Molecular Biotechnology, Faculty of Technology, Bielefeld University, 33615 Bielefeld, Germany
2
Biocatalysis group, Department of Chemistry, Technische Universität Berlin, 10623 Berlin, Germany
3
CO.DON AG, 10587 Berlin, Germany
4
Physical and Biophysical Chemistry (PCIII), Department of Chemistry, Bielefeld University, 33615 Bielefeld, Germany
5
Experimental Biophysics and Applied Nanoscience, Physics Department, Bielefeld University, 33615 Bielefeld, Germany
6
Molecular Biotechnology, Institute for Biochemistry and Biology, University of Potsdam, 14476 Potsdam, Germany
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2019, 20(22), 5702; https://doi.org/10.3390/ijms20225702
Received: 18 September 2019 / Revised: 8 November 2019 / Accepted: 11 November 2019 / Published: 14 November 2019
(This article belongs to the Section Molecular Biology)
Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme β-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with β-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology. View Full-Text
Keywords: adeno-associated-virus; β-lactamase; inverted terminal repeat (ITR); loop modification; capsid stability adeno-associated-virus; β-lactamase; inverted terminal repeat (ITR); loop modification; capsid stability
Show Figures

Graphical abstract

MDPI and ACS Style

Feiner, R.C.; Teschner, J.; Teschner, K.E.; Radukic, M.T.; Baumann, T.; Hagen, S.; Hannappel, Y.; Biere, N.; Anselmetti, D.; Arndt, K.M.; Müller, K.M. rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations. Int. J. Mol. Sci. 2019, 20, 5702.

Show more citation formats Show less citations formats
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop