Construction and Characterization of a High-Capacity Replication-Competent Murine Cytomegalovirus Vector for Gene Delivery
André Riedl,
Denisa Bojková,
Jiang Tan,
Ábris Jeney,
Pia-Katharina Larsen,
Csaba Jeney,
Florian Full,
Ulrich Kalinke,
Zsolt Ruzsics
Affiliations
André Riedl
Medical Center, Institute of Virology, University of Freiburg, 79104 Freiburg, Germany
Denisa Bojková
Medical Center, Institute of Virology, University of Freiburg, 79104 Freiburg, Germany
Jiang Tan
Medical Center, Institute of Virology, University of Freiburg, 79104 Freiburg, Germany
Ábris Jeney
Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany
Pia-Katharina Larsen
TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture between the Hanover Medical School and the Helmholtz Centre for Infection Research, Institute for Experimental Infection Research, 30625 Hanover, Germany
Csaba Jeney
Department of Microsystems Engineering—IMTEK, University of Freiburg, 79110 Freiburg, Germany
Florian Full
Medical Center, Institute of Virology, University of Freiburg, 79104 Freiburg, Germany
Ulrich Kalinke
TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture between the Hanover Medical School and the Helmholtz Centre for Infection Research, Institute for Experimental Infection Research, 30625 Hanover, Germany
Zsolt Ruzsics
Medical Center, Institute of Virology, University of Freiburg, 79104 Freiburg, Germany
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.
