Volume 21, Issue 9, Pages 1738-1748 (September 2013) Meganuclease-mediated Virus Self-cleavage Facilitates Tumor-specific Virus Replication  Engin Gürlevik, Peter Schache, Anneliese Goez, Arnold Kloos, Norman Woller, Nina Armbrecht, Michael P Manns, Stefan Kubicka, Florian Kühnel  Molecular Therapy  Volume 21, Issue 9, Pages 1738-1748 (September 2013) DOI: 10.1038/mt.2013.117 Copyright © 2013 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Concept of specific meganuclease-mediated cleavage of oncolytic adenovirus genomes in nontumor cells. To realize effective cleavage and destruction of oncolytic adenovirus genomes in nontumor cells, recognition sites for the meganuclease I-Sce I are introduced into the virus genome. An additional expression unit provides the virus with a function to respond to intracellular activated p53 by expression of the meganuclease I-Sce I. The figure shows an adenovirus setup as used in this study. (a) In tumor cells harboring dysfunctional p53, the meganuclease is not expressed and viral replication can proceed normally. (b) In normal cells, viral cell entry activates p53 which drives expression of the meganuclease. Subsequent I-Sce I-mediated cleavage of the viral DNA leads to destruction of the adenoviral genome. Molecular Therapy 2013 21, 1738-1748DOI: (10.1038/mt.2013.117) Copyright © 2013 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Genetic setup and functional testing of I-Sce I-expressing oncolytic adenoviruses. (a) To control whether I-Sce-I compromise adenovirus replication, A549 and Huh-7 cells were cotransfected with plasmids coding for I-Sce I, or EGFP as control, and a plasmid for CD90.2-expression for subsequent cell sorting as described in the Methods section. Cells were additionally infected with human wt-Adenovirus serotype 5 at multiplicity of infection (MOI) of 5 to allow for complete infection. The I-Sce I/EGFP expressing subpopulation was isolated by CD90.2-MACS and relative amount of viral genomic DNA was determined by RT-qPCR against the hexon gene locus (EGFP = 1.0; mean + SD). (b) Viral titer in these cells was analyzed by Rapid Titer Assay (mean + SD). (c) Replication-competent, I-Sce I-sensitive adenoviruses were generated for p53-dependent expression of either I-Sce I, or EGFP as nonfunctional control as illustrated in the figure. A minimal version of the hTert promoter flanked by Gal4-binding sites (G) controls expression of the adenoviral E1A gene. In these viruses, the E1 region is flanked by inverted I-Sce I recognition sequences (S). In a second pair of viruses the I-Sce I (EGFP) expression is additionally linked to the p53-dependent expression of the targeted transcriptional repressor GAL4-KRAB (G4K) (d) To characterize the status of transcriptional p53 activity in the cell lines relevant for this study, cells were transfected with a prMinRGC-Luciferase reporter plasmid and pCMV-βgal. After 48 hours, cells were lysed, luceriferase activity was measured, and results were normalized by βgal-activity. (e) p53-positive A549 and HepG2 cells were infected at MOI of 1 with adenoviruses as indicated on top of the panel according to the virus numbering in part Figure 2C. p53-dysfunctional Huh-7 cells were infected at MOI 5 and used as control. Forty hours post infection whole cell extracts were prepared and expression of heterologous proteins was analyzed by western blot. A549 cells infected with Ad-Sce, Ad-EGFP, Ad-G4K-EGFP (from the top) served as exposition control for Huh-7. Molecular Therapy 2013 21, 1738-1748DOI: (10.1038/mt.2013.117) Copyright © 2013 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 I-Sce I expression results in effective cleavage of adenoviral DNA. (a) The figure illustrates double I-Sce I-mediated virus cleavage and PCR-detection of fragments that indicate successful cleavage. After double I-Sce I-mediated cleavage, the E1 gene region is released from the viral backbone. Following religation by DNA damage repair mechanisms the resulting E1-minicircle DNA should be detectable by PCR. (b) Different cell lines were infected with viruses as indicated at an MOI of 0.5 and cellular DNA was harvested 24 hours later. The suggested minicircle DNA (cE1) was detected by PCR and visualized on an agarose gel. Uninfected cells served as control (K). (c) The figure illustrates the design of an additional PCR for identification of both uncleaved and cleaved/religated virus genomes. The results are shown in (d). (e) To detect E1-containing, but cleavage-resistent viruses, 293 cells and A549 cells were infected with adenoviruses at an MOI of 1. After a full replication cycle (48 h), cells were harvested, DNA was extracted and a PCR was performed to amplify a 540 bp fragment containing the distal I-Sce I recognition site (virus plasmid served as control). The resulting fragment was cleaved in vitro with I-Sce I, yielding 300 bp and 240 bp fragments, respectively. F is a prototypic fragment before digestion, M = 1kb marker. Molecular Therapy 2013 21, 1738-1748DOI: (10.1038/mt.2013.117) Copyright © 2013 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Efficient cleavage of oncolytic adenovirus genomes by I-Sce I does not promote DNA damage response-dependent apoptosis. (a) A549 and Huh-7 cells were infected with adenoviruses at MOI 1 in the presence of Doxorubicin (100 ng/ml). Whole cell extracts were prepared and levels of γH2AX were determined by western blot analysis. (b) Induction of apoptosis was tested 36 h post infection (at MOI of 5) by caspase-3 assays. Infection by VSV served as positive control (mean + SD). (c) p53-positive A549 cells were treated with Ad-Sce, or Ad-EGFP, or were left untreated. Tunicamycin was used as positive control. Thirty hours post infection, cells were detached, stained with propidiumiodide (PI) and annexin-V, and were analyzed by FACS. Molecular Therapy 2013 21, 1738-1748DOI: (10.1038/mt.2013.117) Copyright © 2013 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 I-Sce I expression facilitates p53-dependent restriction of adenoviral replication and oncolysis in human cell lines. (a) A549 and Huh-7 cells were infected with indicated viruses (at MOI of 1) in the presence of Doxorubicin (100 ng/ml). Cells were harvested and lysed by repeated freeze–thaw-cycles to release viral particles. Infectious particles were titered by Rapid Titer Assay. (mean + SD). (b) A549 and A549shp53 were infected with indicated viruses at an MOI of 0.05 in the presence of Doxorubicin (100 ng/ml). At the time points indicated, cells were harvested, viral particles were released and titered by Rapid Titer Assay (mean + SD). (c) Cell lines reflecting different p53 activity were infected with adenoviruses and MOIs as indicated. After 7–10 days of infection, cells were fixed and stained with crystal violet. Molecular Therapy 2013 21, 1738-1748DOI: (10.1038/mt.2013.117) Copyright © 2013 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Amplification of I-Sce I-regulated oncolytic adenoviruses is effectively and specifically inhibited in primary human fibroblasts. Primary human IMR-90 fibroblasts were infected with adenovirus at MOI of 0.1. Cells were harvested at time points as indicated and RT-qPCR analysis was performed to investigate specific DNA sequences as noted on top of the diagrams (mean + SD). (a) Detection of the circularized E1 gene region. (b) Detection of the adenoviral hexon gene locus. (c) At different time points as indicated, cells were lysed and the released virus progeny was titered by Rapid Titer Assay. (d) Before adenoviral infection, IMR-90 fibroblasts were transfected twice with siRNA against p53, or with a control siRNA as described in the methods section. Intracellular virus titer was determined. Molecular Therapy 2013 21, 1738-1748DOI: (10.1038/mt.2013.117) Copyright © 2013 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 p53-dependent, I-Sce-mediated self-cleavage of oncolytic adenovirus has limited influence on E1A levels. (a) Cell lines with different p53 status were infected with adenoviruses as described before. After 24 hours of infection, cells were lysed and investigated by western blot analyses. (b) A549 and Huh-7 cells were infected with adenoviruses at an MOI of 0.5. Complete mRNA was harvested at indicated time points. RT-qPCR was performed to determine the relative levels of viral E1A-mRNA (Ad-Sce = 1.0; mean + SD). Molecular Therapy 2013 21, 1738-1748DOI: (10.1038/mt.2013.117) Copyright © 2013 American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Treatment with meganuclease-expressing oncolytic adenoviruses inhibits tumor growth in a p53-dependent manner in murine models of human tumor xenografts. (a) 1 × 107 Huh-7 cells were implanted into the flanks of nude mice to induce growth of tumor xenografts. Once tumors were palpable they were treated with 1 × 109 ifu of oncolytic adenovirus as indicated by intratumoral injection followed by a second injection after one week. Tumor size was monitored (n = 6 per group, ± SD). (b) 1 × 107 A549 (upper panel) or A549shp53 (lower panel) were implanted into the flanks of nude mice. Once tumors had injectable size, mice received an intravenous injection of doxorubicin (17.5 µg/20 g mouse) followed by intravenous administration of 1 × 108 ifu of oncolytic adenovirus as indicated. Tumor size was monitored (n = 6 per group, ± SD). NS, not significant. Molecular Therapy 2013 21, 1738-1748DOI: (10.1038/mt.2013.117) Copyright © 2013 American Society of Gene & Cell Therapy Terms and Conditions