Stable Expression of the Sodium/Iodide Symporter (NIS) for anti-Cancer Gene Therapy of Glioma Cells Using a Third Generation Self-Inactivating Lentiviral Vector System in Combination with 211At
NIS-cDNA (2023bp) was inserted into the backbone vector plasmid pRRLsin.cPPT.CMV.MCS.iresGFP. Virus was produced in 293T using the packaging plasmids pMDLg/pRRE plus pRSV-Rev and the VSV-G envelope-encoding plasmid pMD.G. Transient transfection was performed with Fugene6. Frequency of GFP+ cells was quantified by cell sorter (FACS) and microscopy. NIS expression was examined by immunohistochemistry, iodine and astatine (At, a NISdependent alpha-particle emitting radionuclide) uptake ±perchlorate as specific NIS blocker. Stably NIS-expressing cells were geneticin-selected as positive controls. Experimental therapy measured by clonogenic assay was done using 10/100kBq/ml At-211.
Quantification of viral transduction showed GFP-expression in up to 99.9% of the glioma cells and was clearly higher compared to transient transfection. Mean iodine uptake of transduced cells was 2396cpm/micro gram protein (vs. 2474cpm positive control) and significantly higher than that of transiently transfected cells (469cpm). Iodine uptake was specifically blocked with perchlorate >98% and negative in untransduced controls. At-211 treatment of transduced cells resulted in a specific cell kill.
Robust LV transduction of NIS into target cells resulted in highly stable gene expression and functional activity. Our data suggest that lentiviral vectors are useful tools for NIS gene therapy approaches in combination with alpha or beta emitters.
Keywords: (HIV); 3-3-diaminobenzidine; Clonogenic Assay; DMEM medium; Dulbecco's phosphate buffered saline; Flow Cytometry; Fluorescent Microscopy; Fugene6; GFP-expression; Geneticin; Glioma Cells; Immunostaining; NIS; NIS-cDNA; RPMI16407; Radioiodine therapy; Radionuclides; Rous sarcoma virus; Sodium/Iodide Symporter; Third Generation Self-Inactivating Lentiviral Vector System; Vectastain ABC peroxidase kit; alpha emitter; anti-Cancer Gene Therapy; astatine; central polypurine tract; cyclotron product astatine-211; cytomegalovirus promoter; encephalomyocarditis virus; gag gene; ganciclovir therapy; gene therapy; glioma; human embryonic kidney; human immunodeficiency virus; immunogenicity; internal ribosome entry site; lentivirus; pCIneo-NIS DNA; penicillin; perchlorate; plasmid pRRLsin; radioastatine; streptomycin; viral splice donor
Document Type: Research Article
Publication date: October 1, 2010
- Current Radiopharmaceuticals publishes original research articles, letters, reviews, drug clinical trial studies and guest edited issues on all aspects of research and development of radiolabelled compound preparations. The scope of the journal covers the following areas: radio imaging techniques, therapies; preparation and application of radionuclide compounds including the incorporation of tracer methods used in scientific research and applications.
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