title: Meganuclease-mediated virus self-cleavage facilitates tumor-specific virus replication.
abstract: Meganucleases can specifically cleave long DNA sequence motifs, a feature that makes them an ideal tool for gene engineering in living cells. In a proof-of-concept study, we investigated the use of the meganuclease I-Sce I for targeted virus self-disruption to generate high-specific oncolytic viruses. For this purpose, we provided oncolytic adenoviruses with a molecular circuit that selectively responds to p53 activation by expression of I-Sce I subsequently leading to self-disruption of the viral DNA via heterologous I-Sce I recognition sites within the virus genome. We observed that virus replication and cell lysis was effectively impaired in p53-normal cells, but not in p53-dysfunctional tumor cells. I-Sce I activity led to effective intracellular processing of viral DNA as confirmed by detection of specific cleavage products. Virus disruption did not interfere with E1A levels indicating that reduction of functional virus genomes was the predominant cause for conditional replication. Consequently, tumor-specific replication was further enhanced when E1A expression was additionally inhibited by targeted transcriptional repression. Finally, we demonstrated p53-dependent oncolysis by I-Sce I-expressing viruses in vitro and in vivo, and demonstrated effective inhibition of tumor growth. In summary, meganuclease-mediated virus cleavage represents a promising approach to provide oncolytic viruses with attractive safety profiles.
authors.affiliation: Clinic for Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Carl Neuberg Street 1, 30625 Hannover, Germany.
authors.forename: Engin
authors.initials: E
authors.lastname: Gürlevik
authors.forename: Peter
authors.initials: P
authors.lastname: Schache
authors.forename: Anneliese
authors.initials: A
authors.lastname: Goez
authors.forename: Arnold
authors.initials: A
authors.lastname: Kloos
authors.forename: Norman
authors.initials: N
authors.lastname: Woller
authors.forename: Nina
authors.initials: N
authors.lastname: Armbrecht
authors.forename: Michael P
authors.initials: MP
authors.lastname: Manns
authors.forename: Stefan
authors.initials: S
authors.lastname: Kubicka
authors.forename: Florian
authors.initials: F
authors.lastname: Kühnel
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017500:Adenovirus E1A Proteins / Q000378:metabolism; D002478:Cells, Cultured; D053837:DNA Cleavage; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D056945:Hep G2 Cells; D006801:Humans; D050504:Oncolytic Viruses / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014779:Virus Replication
chemical_list: D017500:Adenovirus E1A Proteins; D004279:DNA, Viral; D029701:Saccharomyces cerevisiae Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific