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. 2024 Jul 18;12(7):791.
doi: 10.3390/vaccines12070791.

Construction and Characterization of a High-Capacity Replication-Competent Murine Cytomegalovirus Vector for Gene Delivery

André Riedl  1   2 , Denisa Bojková  1   2   3 , Jiang Tan  1   2 , Ábris Jeney  2 , Pia-Katharina Larsen  4 , Csaba Jeney  5 , Florian Full  1   2 , Ulrich Kalinke  4 , Zsolt Ruzsics  1   2
Affiliations

Construction and Characterization of a High-Capacity Replication-Competent Murine Cytomegalovirus Vector for Gene Delivery

André Riedl et al. Vaccines (Basel). .

Abstract

We investigated the basic characteristics of a new murine cytomegalovirus (MCMV) vector platform. Using BAC technology, we engineered replication-competent recombinant MCMVs with deletions of up to 26% of the wild-type genome. To this end, we targeted five gene blocks (m01-m17, m106-m109, m129-m141, m144-m158, and m159-m170). BACs featuring deletions from 18% to 26% of the wild-type genome exhibited delayed virus reconstitution, while smaller deletions (up to 16%) demonstrated reconstitution kinetics similar to those of the wild type. Utilizing an innovative methodology, we introduced large genomic DNA segments, up to 35 kbp, along with reporter genes into a newly designed vector with a potential cloning capacity of 46 kbp (Q4). Surprisingly, the insertion of diverse foreign DNAs alleviated the delayed plaque formation phenotype of Q4, and these large inserts remained stable through serial in vitro passages. With reporter-gene-expressing recombinant MCMVs, we successfully transduced not only mouse cell lines but also non-rodent mammalian cells, including those of human, monkey, bovine, and bat origin. Remarkably, even non-mammalian cell lines derived from chickens exhibited successful transduction.

Keywords: cross-species application; cytomegalovirus; gene transfer vector; genetic stability; high-capacity vector; host range.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
(a) The genomic organization of MCMVs (adapted from [26]) features distinct gene blocks. Centrally positioned are the core genes (depicted by black boxes), which exhibit high conservation across herpesvirus families. Adjacent to the core genes are the CMV-specific genes (depicted in light gray and denoted with ‘C’). Towards the termini, homologs shared among the β-herpesviruses are observed (dark gray boxes denoted with ‘β’). Large gene blocks specific to MCMVs are located at the termini (white boxes). To enhance the cargo load capacity, up to five gene blocks of accessory genes (ΔI-V) were selectively deleted. (b) The MCMV vector map illustrates the wild-type MCMV-BAC [47] and the BAC cassette (white box, denoted with ‘BAC’) that was inserted between the deletion clusters II and III to create a circular genome intermediate. The Q4 vector (featuring combined deletions ΔI-IV) and the indicated insertion sites for different site-specific recombinases (open triangles denoted with FRT, loxP, and rox) and an insertion mutant (Q4-LRBAs) were designed to restore the genome size nearly to that of the wild type (the stuffer is indicated by the striped box). The Q4 vector provides a cargo load capacity of almost 46 kbp. Q4-LRBAs represent loaded Q4 vectors with an insert of approximately 37 kbp. (c) Depicted is the result of a multistep growth analysis carried out by infecting MEFs with ΔI+II-GLuc and Q4-LRBAs-GLuc in comparison with the BAC-derived MCMV-wt Smith strain at an MOI of 0.1. Supernatants were collected on the indicated days and titrated using a plaque assay in technical triplicate. The viral titers depicted in (c) represent the results of a single representative experiment. (d) Depicted are multistep growth analyses testing various passages, denoted as P1, P3, and P6, for both the empty MCMV vector Q4 and the cargo-loaded vector Q4-LAD using the BAC-derived MCMV-wt control as described in (c).
Figure 2
Figure 2
Scale representation of four MCMV genomes characterized in detail in this study. Each genome is represented to scale, illustrating the relative sizes and modifications between the wild-type and the engineered variants. For feature explanation, refer to Figure 1. (a) Wild-type MCMV genome (MCMV-wt), comprising 230,277 base pairs (bp). (b) Q4 vector, a deletion variant of the MCMV genome, reduced to 184,892 bp by the removal of gene blocks ΔI-ΔIV. (c) Q4-LAD vector, derived from the Q4 vector, incorporating a large insertion termed LAD, resulting in a total genome size of 218,714 bp. (d) Q4-LRBAs-GLuc vector, similar to Q4-LAD but containing the LRBAs-GLuc insertion instead, with a total genome size of 221,226 bp.
Figure 3
Figure 3
Illumina paired-end sequencing was employed to assess the read coverage of MCMV vectors. DNA extracted from cell-free viruses at designated passages on mouse embryonic fibroblasts (MEFs) served as the sequencing sample. Reads were aligned to reference genomes (see Supplementary Table S3 for the GenBank accession numbers), and deletions were further analyzed for the frequency of deletion reads. Panel (a) illustrates the read coverage of the MCMV-wt genome across various passages. Panels (b–d) represent the read coverage for the empty Q4 vector, Q4-LRBAs-GLuc, and Q4-LAD, respectively.
Figure 4
Figure 4
In vitro transduction efficiency of MCMV vectors in cell lines derived from different species. In panels (a–c), the Gaussian luciferase activity in the supernatant of cultures induced by treatment with the indicated MCMV vectors at an MOI of 3 at 1, 2, and 5 days post-infection (dpi), respectively, is depicted. The luciferase activity was measured indirectly during substrate conversion and is presented here in relative light units (RLUs). Among the transduced cells are fibroblasts of murine, human, and chicken origin, as well as epithelial cells of human, primate, bovine, porcine, canine, bat, and chicken origin. “Mock” represents data from equally treated cells despite viral infection. Data were normalized to the values measured 1 h post-infection (hpi). The values shown are the means of three independent experiments measured in technical triplicate, and the error bars indicate standard deviations. As shown in panels (d,e), the viral genome copies were determined by using M45 gene-specific qPCR at different times after infection of the indicated cell lines with the indicated MCMV vectors. The total DNA extracted from infected MEF, ARPE-19, A549, 293A, 911, and Vero E6 cells was assessed at four different time points (14 hpi, 36 hpi, 3 dpi, and 5 dpi). The resulting viral copy numbers were calculated per haploid genome count, which was determined by using the control qPCR to amplify the GAPDH genes of the respective hosts. (d) The data from three independent experiments are shown pooled together for the MCMV vector ΔI+II-GLuc. (e) Same as (d) for Q4-LRBAs-GLuc.
Figure 5
Figure 5
Release of infectious particles into the supernatant upon infection of various cells with MCMV–wt (a,d) and the MCMV vectors ΔI+II~–GLuc (b,e) and Q4–LRBAs–GLuc (c,f). The indicated cells were infected with the respective viruses at a multiplicity of infection (MOI) of 3 and washed extensively, and the potentially de novo-generated virions were quantified using a standard plaque assay on murine embryonic fibroblasts (MEFs). In the left panels (a–c), cells that produced a detectable titer at 5 dpi or later are shown. In the right panels (d–f), cells that did not produce infectious virions at 5 days post-infection (dpi) or later are depicted. The titers were particularly high in 911 and Vero E6 cells, in addition to the permissive MEFs. The graphs show the results of data from three independent experiments.
Figure 6
Figure 6
Heatmaps depicting viral mRNA transcription in MEF, A549, ARPE-19, and 911 cells following infection with either Q4-LRBAs-GLuc (Vec) or wild-type MCMV (WT) at early (8 h post-infection (hpi)) and late (31 hpi) time points. The log2 of normalized read counts by counts per million (cpm) is presented as the MCMV transcript level. Each column corresponds to an individual experiment (mock experiments are split into two replicates). The genes coding for Q4-LRBAs-GLuc are presented. The expression of each gene has been scaled. Samples with relatively high expression of an MCMV gene are marked in red, and samples with relatively low expression are marked in yellow and blue. White areas represent genes with no counts.
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