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. 2024 Apr 23;16(5):658.
doi: 10.3390/v16050658.

Expanding the Scope of Adenoviral Vectors by Utilizing Novel Tools for Recombination and Vector Rescue

Julian Fischer  1 , Ariana Fedotova  1 , Clara Bühler  1 , Laura Darriba  1 , Sabrina Schreiner  1 , Zsolt Ruzsics  1
Affiliations

Expanding the Scope of Adenoviral Vectors by Utilizing Novel Tools for Recombination and Vector Rescue

Julian Fischer et al. Viruses. .

Abstract

Recombinant adenoviruses are widely used in clinical and laboratory applications. Despite the wide variety of available sero- and genotypes, only a fraction is utilized in vivo. As adenoviruses are a large group of viruses, displaying many different tropisms, immune epitopes, and replication characteristics, the merits of translating these natural benefits into vector applications are apparent. This translation, however, proves difficult, since while research has investigated the application of these viruses, there are no universally applicable rules in vector design for non-classical adenovirus types. In this paper, we describe a generalized workflow that allows vectorization, rescue, and cloning of all adenoviral species to enable the rapid development of new vector variants. We show this using human and simian adenoviruses, further modifying a selection of them to investigate their gene transfer potential and build potential vector candidates for future applications.

Keywords: human adenoviruses; mutagenesis of viral genomes; replication-competent vectors; transgene insertion sites; viral bacmids.

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Conflict of interest statement

J.F. and Z.R. are co-inventors in the patent application EP23199021.9 by the Albert-Ludwigs-University of Freiburg, which describes a two-step workflow to seamlessly modify circular BAC-/plasmids at high efficiency. J.F. and Z.R. are co-inventors in the patent application PCT/EP2021/076757 by the Albert-Ludwigs-University of Freiburg, which describes a novel way of generating recombinant Adenoviruses by utilizing CRISPR/Cas9 linearization.

Figures

Figure A1
Figure A1
Schematic overview of HFR workflow for transgene insertion into replicating vectors on the example of Insertion site I1. (a) Target sites are determined by proximally occurring half sites of the target enzyme (SwaI; ATTT/AAAT) and region between half sites replaced with kanamycin resistance cassette (KnR) using red recombination. (b) Resulting intermediate construct can be selected via KnR and digested using SwaI, as KnR also completed restriction sites. (c) Linear insert carrying transgene cassette (TG) and the removed wild-type sequences can be combined with linearized bacmids from the previous step. (d) Final TG-carrying bacmid contains TG is otherwise completely unaffected, resulting in the scarless assembly of recombinant bacmids.
Figure 1
Figure 1
Assembly of bacmids carrying AdV genomic DNA. (a) Schematic overview of bacmid assembly, starting with genomic DNA (blue) extraction from viral particles. The plasmid backbone (orange arrow) is amplified from pKSB2 via PCR, adding ACT sequences (yellow boxes) and homologies to viral ITR (green) as well as internal overlaps between PCR fragments (orange boxes). Fragments are combined with DNA using Gibson assembly. The finished assembly products can incorporate genomic DNA in 2 orientations, either (b) with E1A (red arrow) and backbone coding in the same direction or (c) with E1A and backbone coding in opposite directions.
Figure 2
Figure 2
sgRNAs used for CTR. Schematic view of ACT sequences (yellow) and adjacent ITR (green/magenta) and bacmid (orange) sequences present on genomic bacmids carrying the indicated HAdV genomes. The targeted sites of the generally applicable (sgRNA-Ex) and the more specific (sgRNA-Int4 and -Int5) sgRNAs used for CTR are shown below or above the sequences, respectively, with arrows indicating cutting sites. PAMs are underlined.
Figure 3
Figure 3
Generation of replication-competent reporter virus vectors. (a) Schematic view of HAdV genome, with the target sites for transgenic insertion indicated. Early genetic regions are marked red, late genetic regions in green. Transgenic viruses were based on E3-deleted variants to make appropriate space for transgene cassette; (b) Schematic view of inserted transgene cassette; MIE: MCMV immediate early promoter; GFP: Green fluorescent protein; GLuc: Gaussia luciferase; CI: Chimeric intron cloned from pMT2; SV40pA: Poly-A tail of SV40.
Figure 4
Figure 4
Expression of transgenes over time as determined by a Luciferase assay for (a) HAdV-B3-based recombinants and (b) HAdV-E4-based recombinants. Area under the curve for luciferase measurements of (a,b), displayed for easier comparison for (c) HAdV-B3-based recombinants and (d) HAdV-E4-based recombinants. Statistical significance was determined by an ordinary one-way ANOVA with Tukeys’s multiple comparisons. Significance is indicated as follow: *** p < 0.0001, and ns: not significant.
Figure 5
Figure 5
(a) Fluorescent microscopy of representative plaques of different recombinant viruses. Contrast is boosted equally across all pictures for better clarity. Scale bar represents 200 μm. Growth curves for recombinant viruses compared to wild-type and non-transgenic E3-deleted construct for (b) HAdV-B3 and (c) HAdV-E4. Dashed line represents lower detection limit. Statistical significance was determined against the corresponding wild-type virus. Statistical significance was determined by an ordinary two-way ANOVA. Significance is indicated as follow: * p < 0.01.
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