Construction and expression in tumor cells of a recombinant vaccinia virus encoding human interleukin-1 beta.
BACKGROUND
Human interleukin-1 beta (hIL-1 beta) injected intratumorally has demonstrated growth inhibition of transplanted subcutaneous tumors in mice, regression of metastatic lesions, resistance to tumor rechallenge, and increased survival. Vaccinia virus (VV) can be genetically engineered to produce cytokines and may be an effective vector for gene therapy of cancer. This study was designed to (a) construct a VV expressing hIL-1 beta, (b) assess tumor cell infection in vitro with this construct, (c) measure hIL-1 beta production, and (d) assess the bioactivity of the secreted cytokine.


METHODS
The hIL-1 beta gene was amplified from a plasmid clone using polymerase chain reaction (PCR) and then cloned into a homologous recombination (HR) and expression vector, which was used to insert the hIL-1 beta gene into the VV genome. Selection of the recombinant VV (vMJ601hIL-1 beta) was based on inactivation of viral TK and expression of beta-galactosidase. vMJ601hIL-1 beta infectivity and cytokine production was assessed by infecting tumor cell lines and analyzing culture supernatants for hIL-1 beta. Bioactivity of the hIL-1 beta produced was demonstrated using an IL-1 dependent T helper cell line.


RESULTS
The hIL-1 beta gene was successfully cloned into the VV genome by HR, which was confirmed by PCR. vMJ601hIL-1 beta efficiently infected tumor cells, as shown by increased hIL-1 beta secretion (0 to > 500 ng/ml) and morphologic evidence of viral cytopathic effect. vMJ601hIL-1 beta-infected cells secreted large amounts of hIL-1 beta (mean 772 ng/10(6) cells/24 h). The secreted hIL-1 beta was bioactive (mean bioactivity 6.8 x 10(8) U/mg of hIL-1 beta).


CONCLUSIONS
(a) hIL-1 beta can be cloned into VV, (b) vMJ601hIL-1 beta retains its infectivity, (c) a large amount of hIL-1 beta is secreted, and (d) the secreted hIL-1 beta is bioactive. Recombinant VV may allow in situ cytokine gene delivery and expression in established tumors.
Peplinski
G R
GR
Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Tsung
K
K
Whitman
E D
ED
Meko
J B
JB
Norton
J A
JA
D000818:Animals; D001682:Biological Availability; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D005240:Feasibility Studies; D015972:Gene Expression Regulation, Neoplastic; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D009374:Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D011995:Recombination, Genetic; D006377:T-Lymphocytes, Helper-Inducer / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus / Q000235:genetics* / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004279:DNA, Viral; D007375:Interleukin-1; D001616:beta-Galactosidase