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

  • 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 beta-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 beta-lactamase allowed for theRecombinant 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 beta-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 beta-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.show moreshow less

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Metadaten
Author:Rebecca Christine FeinerORCiDGND, Julian TeschnerORCiDGND, Kathrin E. TeschnerGND, Marco T. Radukic, Tobias BaumannGND, Sven HagenORCiD, Yvonne HannappelORCiD, Niklas BiereORCiD, Dario AnselmettiORCiDGND, Katja Maren ArndtORCiDGND, Kristian Mark MüllerGND
DOI:https://doi.org/10.3390/ijms20225702
ISSN:1422-0067
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=31739438
Parent Title (English):International journal of molecular sciences
Publisher:MDPI
Place of publication:Basel
Document Type:Article
Language:English
Year of first Publication:2019
Year of Completion:2019
Release Date:2020/10/11
Tag:adeno-associated-virus; beta-lactamase; capsid stability; inverted terminal repeat (ITR); loop modification
Volume:20
Issue:22
Pagenumber:19
Funder:German Research Foundation (DFG)German Research Foundation (DFG); Bielefeld University
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Peer Review:Referiert
Publication Way:Open Access
Open Access / Gold Open-Access
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