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A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA
Published on: December 26, 2015
Receptor-mediated delivery of an antisense gene to human brain cancer cells
Yun Zhang1, Hwa Jeong Lee, Ruben J Boado
1Department of Medicine, UCLA School of Medicine, Los Angeles, CA 90024, USA.
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.
Conclusion:
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.
Insights
Pegylated immunoliposomes (PILs) deliver an epidermal growth factor receptor (EGFR) antisense gene to brain cancer cells, inhibiting growth by 70-80%. This non-viral gene targeting technology enables in vivo delivery via intravenous administration.
Area of Science:
- Oncology
- Gene Therapy
- Nanotechnology
Background:
- High-grade brain gliomas overexpress the epidermal growth factor receptor (EGFR).
- EGFR antisense gene therapy offers a potential strategy to inhibit the growth of EGFR-dependent gliomas.
- Development of effective in vivo gene delivery systems for brain cancer is crucial.
Purpose of the Study:
- To develop a novel gene targeting technology for delivering therapeutic genes to brain cancer cells.
- To utilize pegylated immunoliposomes (PILs) for in vivo delivery of an EGFR antisense gene following intravenous administration.
- To assess the efficacy of PIL-mediated gene delivery and its impact on glioma cell growth.
Main Methods:
- Constructed a human EGFR antisense gene driven by the SV40 promoter in a non-viral plasmid.
- Packaged the plasmid into 85 nm pegylated immunoliposomes (PILs).
- Targeted PILs to U87 human glioma cells using a murine monoclonal antibody (MAb) to the human insulin receptor (HIR).
Main Results:
- Confocal microscopy confirmed internalization of PILs and nuclear entry of delivered genes in glioma cells.
- PILs successfully delivered exogenous genes to nearly all cultured cells, achieving luciferase expression comparable to lipofectamine.
- Targeting the EGFR antisense gene via PILs resulted in a >70% reduction in [(3)H]thymidine incorporation and a 79% decrease in EGFR levels.
Conclusions:
- Demonstrated successful targeting of an EGFR antisense gene to human brain cancer cells using PILs.
- Achieved significant inhibition (70-80%) of brain cancer cell growth.
- PILs represent a promising non-viral vector for effective in vivo gene targeting via intravenous administration.
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