Receptor-mediated delivery of an antisense gene to human brain cancer cells.
BACKGROUND
The goal of this work was the development of a gene targeting technology that will enable the delivery of therapeutic genes to brain cancer cells in vivo following intravenous administration. High-grade brain gliomas overexpress the epidermal growth factor receptor (EGFR) and EGFR antisense gene therapy could reduce the growth of EGFR-dependent gliomas.


METHODS
A human EGFR antisense gene driven by the SV40 promoter in a non-viral plasmid carrying elements that facilitate extra-chromosomal replication was packaged in the interior of 85 nm pegylated immunoliposomes (PILs). The PILs were targeted to U87 human glioma cells with the 83-14 murine monoclonal antibody (MAb) to the human insulin receptor (HIR).


RESULTS
Confocal fluorescent microscopy demonstrated that the unconjugated HIR MAb is rapidly internalized by the glioma cells. Endocytosis followed by entry into the nucleus was also demonstrated for the HIR MAb conjugated PILs carrying fluorescein-labeled plasmid DNA. The PILs delivered exogenous genes to virtually all cells in culture, based on beta-galactosidase histochemistry. The targeting of a luciferase gene to the U87 cells with the PILs resulted in luciferase levels in excess of 150 pg/mg protein after 72 h of incubation. The level of luciferase gene expression in the U87 cells achieved with the PIL gene targeting system was comparable to that with lipofectamine. Targeting the EGFR antisense gene to U87 glioma cells with the PILs resulted in more than 70% reduction in [(3)H]thymidine incorporation into the cells; this was paralleled by a 79% reduction in the level of immunoreactive EGFR.


CONCLUSIONS
The present work describes the targeting of an EGFR antisense gene to human brain cancer cells, which results in a 70-80% inhibition in cancer cell growth. PILs provide a new approach to gene targeting that is effective in vivo following intravenous administration without viral vectors.
Zhang
Yun
Y
Department of Medicine, UCLA School of Medicine, Los Angeles, CA 90024, USA.
Jeong Lee
Hwa
H
Boado
Ruben J
RJ
Pardridge
William M
WM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001812:Blood-Brain Barrier; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism / Q000628:therapy*; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000235:genetics; D019793:Fluorescein / Q000494:pharmacology; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D008081:Liposomes; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D004247:DNA; D008156:Luciferases; D066246:ErbB Receptors; D001616:beta-Galactosidase; D019793:Fluorescein; D013936:Thymidine