Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes.
We have demonstrated previously the oncolytic effects of a systemically delivered, replicating vaccinia virus. To enhance the tumor specificity of this vector, we have developed a combined thymidine kinase-deleted (TK-) and vaccinia growth factor-deleted (VGF-) vaccinia virus and investigated its properties in vitro and in vivo. The gene for enhanced green fluorescent protein (EGFP) was inserted into the TK locus of a VGF- vaccinia virus by homologous recombination creating a double-deleted mutant vaccinia virus (vvDD-GFP). Infection of resting and dividing NIH3T3 cells with vvDD-GFP yielded reduced viral recovery compared with wild-type (WT), TK-, or VGF- viruses from resting cultures but equivalent virus recovery from dividing cultures. Eight days after nude mice were injected i.p. with 10(7) plaque-forming units (pfu) of WT, TK-, VGF-, or vvDD-GFP vaccinia virus, tissues and tumor were harvested for viral titer determination. No virus was recovered from the brains of mice injected with vvDD-GFP compared with the other viruses, which ranged from 130 to 28,000 pfu/mg protein; however, equivalent amounts were recovered from tumor. There was no toxicity from vvDD-GFP because nude mice receiving 10(8) pfu of IP vvDD-GFP lived >100 days, whereas mice receiving WT, VGF-, or TK- virus had median survivals of only 6, 17, and 29 days, respectively. Similar results were seen when 10(9) pfu of vvDD-GFP were given. Nude mice bearing s.c. murine colon adenocarcinoma (MC38) had significant tumor regression after treatment with 10(9) pfu of systemic (i.p.) vvDD-GFP compared with control (mean tumor size, 180.71 +/- 35.26 mm(3) versus 2796.79 +/- 573.20 mm(3) 12 days after injection of virus). Our data demonstrate that a TK- and VGF- mutant vaccinia virus is significantly attenuated in resting cells in vitro and demonstrates tumor-specific replication in vivo. It is a promising vector for use in tumor-directed gene therapy, given its enhanced safety profile, tumor selectivity, and the oncolytic effects after systemic delivery.
McCart
J A
JA
Surgery Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892-1502, USA.
Ward
J M
JM
Lee
J
J
Hu
Y
Y
Alexander
H R
HR
Libutti
S K
SK
Moss
B
B
Bartlett
D L
DL
D016475:3T3 Cells; D000818:Animals; D003588:Cytopathogenic Effect, Viral; D005260:Female; D017353:Gene Deletion; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D000882:Haplorhini; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D010455:Peptides / Q000235:genetics*; D011213:Poxviridae Infections / Q000821:virology; D013937:Thymidine Kinase / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D014779:Virus Replication
D036341:Intercellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D010455:Peptides; C051087:vaccinia growth factor; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase