Gene therapy for malignant glioma using Sindbis vectors expressing a fusogenic membrane glycoprotein.
BACKGROUND
Malignant glioma has a dismal prognosis. It was previously shown that glioma cells are efficiently killed when they express a gene coding for a hyperfusogenic mutant of the gibbon ape leukemia virus envelope glycoprotein (GALV.fus). However, production of viral vectors expressing GALV.fus has proven problematic because the transgene is toxic to vector-producing cells of human origin. We reasoned that Sindbis-virus-based vectors might be ideal for GALV.fus gene transfer because high-titer stocks can easily be generated in hamster cells and Sindbis virus efficiently infects human tumor cells through the high-affinity 67 kDa laminin receptor. In addition, Sindbis virus nonstructural proteins are potent inducers of apoptosis, and Sindbis vector RNAs expressing fusogenic viral proteins have been shown to spread from cell-to-cell in membrane-formed infectious particles.


METHODS
Sindbis virus replicon-containing particles were generated by co-transfecting vector and helper RNAs into baby hamster kidney (BHK-21) cells. Packaged beta-galactosidase and GALV.fus expressing Sindbis vectors were used to infect glioma cell lines, which were then compared for syncytial cytopathic effect, cell killing, and release of infectious virus-like particles containing the vector genome. Finally, the efficacy of GALV.fus and beta-galactosidase Sindbis vectors was compared in an orthotopic intracerebral U87 glioma xenograft model in nude mice.


RESULTS
High-titer stocks (>10(9) infectious units (iu)/ml) of the GALV.fus and beta-galactosidase vectors were obtained. Glioma cells infected with the GALV.fus vector formed large syncytia which died rapidly by apoptosis and released infectious membrane-formed particles that could transfer vector genomes to uninfected cells. The GALV.fus vector had significantly greater antitumor therapeutic potency than the beta-galactosidase vector in the U87 glioma xenograft model.


CONCLUSIONS
Sindbis vectors expressing GALV.fus can be packaged into infectious viral particles to high titer, they exhibit potent bystander cytopathic potential and are active against U87 glioma xenografts. Sindbis-virus-based replicons appear to be efficient vector systems for delivery and expression of fusogenic membrane glycoproteins.
Zhang
Jie
J
Mayo Clinic, Molecular Medicine Program, 200 First Street, SW, Rochester, MN 55905, USA.
Frolov
Ilya
I
Russell
Stephen J
SJ
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001932:Brain Neoplasms / Q000628:therapy; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D053938:DNA Fragmentation; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D005910:Glioma / Q000628:therapy*; D006801:Humans; D020287:In Situ Nick-End Labeling; D007763:Lac Operon; D008562:Membrane Glycoproteins / Q000737:chemistry*; D051379:Mice; D008819:Mice, Nude; D008957:Models, Genetic; D009368:Neoplasm Transplantation; D010957:Plasmids / Q000378:metabolism; D012845:Sindbis Virus / Q000235:genetics*; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
D000970:Antineoplastic Agents; D008562:Membrane Glycoproteins; D001616:beta-Galactosidase