Volume 20, Issue 5, Pages 994-1001 (May 2012) Replication-competent Herpes Simplex Virus Retargeted to HER2 as Therapy for High- grade Glioma  Eleonora Gambini, Elisa Reisoli, Irene Appolloni, Valentina Gatta, Gabriella Campadelli-Fiume, Laura Menotti, Paolo Malatesta  Molecular Therapy  Volume 20, Issue 5, Pages 994-1001 (May 2012) DOI: 10.1038/mt.2012.22 Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 R-LM113 is safe in vivo. Merged fluorescence and bright-field images of adult nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse brains after injection of (a–c) 105 plaque-forming unit (pfu) R-LM5 or (f–h) 3 × 105 pfu R-LM113. Viral spread is visualized by enhanced green fluorescent protein fluorescence. Coronal sections of adult NOD/SCID mouse brains after injection of (d,e) R-LM5 or (i,j) R-LM113, stained with (d,i) hematoxylin and eosin or (e, j) antibody for the indicated markers. To note, the presence of extravasation areas (hollow arrowheads in d) and infiltration of granulocytes (arrowheads in e) and macrophages (arrow in e) in the parenchyma of R-LM5-injected brains. On the contrary, granulocytes are exclusively found inside blood vessels of R-LM113-injected brains (arrowheads in j). Bar = 20 µm. H&E, hematoxylin and eosin; MPO, myeloperoxidase. Molecular Therapy 2012 20, 994-1001DOI: (10.1038/mt.2012.22) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 HER2 expression in mGBM cells does not alter their characteristics. (a–h) Immunofluorescence stainings of (a–d) mGBM and (e–h) mGBM-HER2 cultured cells for the indicated markers in green. (j) Cumulative labeling analysis using EdU was performed to compare the growth fraction, the length of the cell cycle, and the length of the S phase of mGBM (red) and mGBM-HER2 (green) cells. Cumulative labeling increases linearly depending on the length of the cell cycle and S phase, and show a plateau when all cycling cells are labeled. The regression lines for the five points preceding the plateau (up to 10 hours) and their respective confidence intervals (shadings) are represented with the same color code. Of note, the confidence interval for mGBM-HER2 lies inside that of mGBM cells, showing that their cell cycle parameters are indistinguishable. Subsequent time points (24, 30, and 34 hours) are included in the plot in order to show the plateau of the labeling indexes (also known as growth fractions). (k) Merged fluorescence and bright-field images of a typical mGBM-HER2-derived tumor in dorsal view. The tumor is detectable as DsRed expression. (l–u) Coronal sections of tumors derived by mGBM-HER2 cells stained with hematoxylin and eosin (l,m) or with the indicated antibody (n–u). The dashed line in l defines the contour of a pseudopalisade; the arrowhead in m indicates area of extravasation. Arrows indicate macrophages in n; arrowheads indicate granulocytes in n and o. DsRed endogenous fluorescence of tumor cells is visible in red in p–u. Nuclei are counterstained in blue with Hoechst 33342 in a–h, n–u. Bar = 15 µm (a and e); 25 µm (b–d, f–h, m); 1.2 mm (k); 40 µm (l, p–u); 8 µm (n and o). GFAP, glial fibrillary acidic protein; H&E, hematoxylin and eosin; mGBM, murine glioblastoma; MPO, myeloperoxidase. Molecular Therapy 2012 20, 994-1001DOI: (10.1038/mt.2012.22) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 The recombinant virus R-LM113 is able to infect solely mGBM-HER2 cells. (a–d′) Matched micrographs showing (a–d) bright-field and (a′–d′) virus-encoded EGFP fluorescence of (a,b) mGBM or (c,d) mGBM-HER2 cells 24 hours after exposure to (a,c) R-LM113 or (b,d) R-LM5 at multiplicity of infection (MOI) = 5. (e–f) Representative dot plots of mGBM-HER2 cells exposed to (e) R-LM113 or (f) R-LM5. Numbers in quadrants indicate the percentage of infected cells evaluated as EGFP expression. Lines correspond to the thresholds used. (g–i) Cell-to-cell spread of the R-LM113 recombinant virus in mGBM-HER2 at different time points after infection with MOI = 0.025. Bar = 50 µm. EGFP, enhanced green fluorescent protein. Molecular Therapy 2012 20, 994-1001DOI: (10.1038/mt.2012.22) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 R-LM113 counteracts the growth of HER2-expressing gliomas. (a) Kaplan–Meier plot for animals transplanted with mGBM-HER2 cells alone (red line: early-treatment control set) or together with R-LM113-infected mGBM-HER2 (green line: R-LM113 early-treatment set). The crosses indicate mice censored for subsequent analysis. (b) Merged DsRed fluorescence and bright-field image of a brain from a mouse of the control set (arrowhead labeled as b* in a). (c) Coronal section of the brain from a mouse of the R-LM113 early-treatment set censored 66 days post-transplant (arrowhead labeled as c* in a); in green is shown enhanced green fluorescent protein expressed from the virus, in blue the nuclei. (d) Merged red and green fluorescence and bright-field images of the brain from a mouse of the R-LM113 early-treatment set censored 161 days post-transplant (arrowhead labeled as d* in a). (d′) Coronal section of the brain shown in d, along the dashed line. mGBM, murine glioblastoma. Molecular Therapy 2012 20, 994-1001DOI: (10.1038/mt.2012.22) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 R-LM113 improves mouse survival when injected in already established HER2-expressing gliomas. (a) Kaplan–Meier plot for animals bearing mGBM-HER2 tumors inoculated with R-LM113 (green line) or with ultraviolet-inactivated R-LM113 (red line). (b,c) Merged red and green fluorescence and bright-field images of brains from mice of the R-LM113 late-treatment set died at 6 days (b, arrowhead labeled as b* in panel a) and at 37 days (c, arrowhead labeled as c* in panel a) following R-LM113 inoculation. (d) Coronal section of a mGBM-HER2-bearing mouse injected with R-LM113 and killed 5 days postinoculation. In green is shown the virally expressed enhanced green fluorescent protein, in red the endogenous fluorescence of DsRed expressed by mGBM-HER2, in blue the nuclei. The right inset shows a magnification of the region distant ∼1.3 mm. AON accessory olfactory nucleus, CTX, cortex; HSV, herpes simplex virus; rf, rhinal fissure. Molecular Therapy 2012 20, 994-1001DOI: (10.1038/mt.2012.22) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions