Tumor Growth Inhibition by Intratumoral Inoculation of Defective Herpes Simplex Virus Vectors Expressing Granulocyte–Macrophage Colony-Stimulating Factor  Masahiro Toda, Robert L. Martuza, Samuel D. Rabkin  Molecular Therapy  Volume 2, Issue 4, Pages 324-329 (October 2000) DOI: 10.1006/mthe.2000.0130 Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 1 Structure of the amplicon plasmids. (A) pHCGM-tk contains murine GM-CSF cDNA under control of the CMVIE promoter and SV40 polyadenylation sequence (SV40 polyA). (B) pHCL-tk contains the E. coli lacZ gene under control of the CMVIE promoter and SV40 polyadenylation sequence. The amplicon plasmids also contain: HSV cis-acting sequences HSV a, the HSV-1 cleavage/packaging signal, and HSV ori, the HSV-2 origin of DNA replication; HSV tk under control of the HSV-1 tk promoter; and bacterial plasmid sequences β-lactamase (AmpR, ampicillin resistance) and the plasmid origin of DNA replication (ColE Molecular Therapy 2000 2, 324-329DOI: (10.1006/mthe.2000.0130) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 2 Synthesis of murine GM-CSF by various mouse tumor cell lines and rat cerebellar granule cells (Neuron) infected with dvGM/tsK at a m.o.i. of 1 (of tsK). Culture supernatants were collected after 48 h and assayed for murine GM-CSF using a sandwich ELISA. Molecular Therapy 2000 2, 324-329DOI: (10.1006/mthe.2000.0130) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 3 Effect of dvlacZ/tsK and dvGM/tsK infection on Harding–Passey cell growth in vitro. Monolayer cell cultures were infected with defective HSV vectors at a m.o.i. of 0.1 (of tsK). Viable, trypan blue-excluding cells were counted on the days postinfection indicated. Data plotted are means of duplicate wells. Molecular Therapy 2000 2, 324-329DOI: (10.1006/mthe.2000.0130) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 4 Bilateral established sc mouse melanoma tumor therapy. When bilateral sc tumors reached approximately 5 mm in maximal diameter, mice underwent unilateral intratumoral inoculation with defective HSV vectors (2 × 103 to 2 × 105 pfu of tsK) or virus buffer (Mock) into the right side tumor on day 0 (n = 5/group). (A) Growth of inoculated (Rt) and noninoculated (Lt) sc tumors after inoculation. (B) Tumor volumes were compared on day 14 postinoculation. Both the inoculated tumors (Rt) and their noninoculated contralateral counterparts (Lt) demonstrated significant inhibition of tumor growth after inoculation with dvGM/tsK (2 × 105 pfu of tsK), compared to dvlacZ/tsK [P < 0.02 (Rt), P < 0.05 (Lt), unpaired t test] and mock [P < 0.02 (Rt), P < 0.02 (Lt)]. Bars represent means ± SEM. *P < 0.05, unpaired t test. dvlacZ (5), dvlacZ/tsK (2 × 105 pfu of tsK); dvGM (5), dvGM/tsK (2 × 105 pfu of tsK); dvGM (4), dvGM/tsK (2 × 104 pfu of tsK); dvGM (3), dvGM/tsK (2 × 103 pfu o Molecular Therapy 2000 2, 324-329DOI: (10.1006/mthe.2000.0130) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 5 Survival of tumor-bearing mice treated with defective HSV vectors. When bilateral subcutaneous tumors reached approximately 5 mm in maximal diameter, mice underwent unilateral intratumoral inoculation with defective HSV vectors (2 × 103 to 2 × 105 pfu of tsk) or virus buffer (Mock) into the right side tumor on day 0 (n = 5/group) (experiment in Fig. 4). When mice became moribund or tumors reached >18 mm in diameter they were sacrificed. Mean survival of dvGM/tsK (2 × 105 pfu; 40.4 ± 11.9) was significantly different from that of lower dose dvGM/tsK (2 × 104 pfu; 28.2 ± 3.0 and 2 × 103 pfu; 26.2 ± 3.2), dvlacZ/tsK (27.8 ± 4.5), and mock (25.6 ± 3.9) (Fisher's pairwise least significant difference, P < 0.05). Survival of mice treated with dvGM/tsK (2 × 105 pfu) was significantly greater than mice treated with dvGM/tsK (2 × 103 pfu), dvlacZ/tsK, and mock (P < 0.05, Mantel–Co Molecular Therapy 2000 2, 324-329DOI: (10.1006/mthe.2000.0130) Copyright © 2000 American Society for Gene Therapy Terms and Conditions