We recently demonstrated tumor-selective iodide uptake and therapeutic efficacy of radioiodine in neuroblastoma tumors after systemic nonviral polyplex-mediated sodium iodide symporter (NIS) gene delivery. In the present study, we used novel polyplexes based on linear polyethylenimine (LPEI), polyethylene glycol (PEG), and the synthetic peptide GE11 as an epidermal growth factor receptor (EGFR)-specific ligand to target a NIS-expressing plasmid to hepatocellular carcinoma (HCC) (HuH7). Incubation of HuH7 cells with LPEI-PEG-GE11/NIS polyplexes resulted in a 22-fold increase in iodide uptake, which was confirmed in other cancer cell lines correlating well with EGFR expression levels. Using (123)I-scintigraphy and ex vivo γ-counting, HuH7 xenografts accumulated 6.5-9% injected dose per gram (ID/g) (123)I, resulting in a tumor-absorbed dose of 47 mGray/Megabecquerel (mGy/MBq) (131)Iodide ((131)I) after intravenous (i.v.) application of LPEI-PEG-GE11/NIS. No iodide uptake was observed in other tissues. After pretreatment with the EGFR-specific antibody cetuximab, tumoral iodide uptake was markedly reduced confirming the specificity of EGFR-targeted polyplexes. After three or four cycles of polyplex/(131)I application, a significant delay in tumor growth was observed associated with prolonged survival. These results demonstrate that systemic NIS gene transfer using polyplexes coupled with an EGFR-targeting ligand is capable of inducing tumor-specific iodide uptake, which represents a promising innovative strategy for systemic NIS gene therapy in metastatic cancers.

Due to its dual role as reporter and therapy gene, the sodium iodide symporter (NIS) allows noninvasive imaging of functional NIS expression by (123)I-scintigraphy or (124)I-PET imaging before the application of a therapeutic dose of (131)I. NIS expression provides a novel mechanism for the evaluation of mesenchymal stem cells (MSCs) as gene delivery vehicles for tumor therapy. In the current study, we stably transfected bone marrow-derived CD34(-) MSCs with NIS cDNA (NIS-MSC), which revealed high levels of functional NIS protein expression. In mixed populations of NIS-MSCs and hepatocellular cancer (HCC) cells, clonogenic assays showed a 55% reduction of HCC cell survival after (131)I application. We then investigated body distribution of NIS-MSCs by (123)I-scintigraphy and (124)I-PET imaging following intravenous (i.v.) injection of NIS-MSCs in a HCC xenograft mouse model demonstrating active MSC recruitment into the tumor stroma which was confirmed by immunohistochemistry and ex vivo γ-counter analysis. Three cycles of systemic MSC-mediated NIS gene delivery followed by (131)I application resulted in a significant delay in tumor growth. Our results demonstrate tumor-specific accumulation and therapeutic efficacy of radioiodine after MSC-mediated NIS gene delivery in HCC tumors, opening the prospect of NIS-mediated radionuclide therapy of metastatic cancer using MSCs as gene delivery vehicles.

Due to limited treatment options the prognosis of patients with advanced hepatocellular cancer (HCC) has remained poor. To investigate an alternative therapeutic approach, we examined the feasibility of radioiodine therapy of HCC following human sodium iodide symporter (NIS) gene transfer using a mouse a-fetoprotein (AFP) promoter construct to target NIS expression to HCC cells. For this purpose, the murine Hepa 1-6 and the human HepG2 hepatoma cell lines were stably transfected with NIS cDNA under the control of the tumor-specific AFP promoter. The stably transfected Hepa 1-6 cell line showed a 10-fold increase in iodide accumulation, while HepG2 cells accumulated 125 I approximately 60-fold. Tumor-specific NIS expression was confirmed on mRNA level by northern blot analysis, and on protein level by immunostaining, that revealed primarily membrane-associated NIS-specific immunoreactivity.In an in vitro clonogenic assay up to 78% of NIS-transfected Hepa 1-6 and 93% of HepG2 cells were killed by

Purpose: We recently reported the significant therapeutic efficacy of radioiodine therapy in various tumor mouse models following transcriptionally targeted sodium iodide symporter (NIS) gene transfer. These studies showed the high potential of NIS as a novel diagnostic and therapeutic gene for the treatment of extrathyroidal tumors. As a next crucial step towards clinical application of NIS-mediated radionuclide therapy we aim at systemic delivery of the NIS gene to target extrathyroidal tumors even in the metastatic stage. Experimental Design: In the current study, we used synthetic polymeric vectors based on pseudodendritic oligoamines with high intrinsic tumor affinity (G2-HD-OEI) to target a NIS-expressing plasmid (CMV-NIS-pcDNA3) to neuroblastoma (Neuro2A) cells. Results: Incubation with NIS-containing polyplexes (G2-HD-OEI/NIS) resulted in a 51-fold increase in perchlorate-sensitive iodide uptake activity in Neuro2A cells in vitro. The exact mechanism by which iodide is actively transported across the basolateral membrane of thyroid follicular cells was clarified by the cloning and characterization of the sodium iodide symporter (NIS) 13 years ago (1-3). NIS, an intrinsic transmembrane glycoprotein with 13 putative transmembrane domains, is responsible for the ability of the thyroid gland to concentrate iodide, the first and rate-limiting step in the process of thyroid hormonogenesis (4, 5). Moreover, due to its expression in follicular cell-derived thyroid cancer cells, NIS provides the molecular basis for the diagnostic and therapeutic application of radioiodine, which has been successfully used for more than 60 years in the treatment of thyroid cancer patients and therefore represents the most effective form of systemic anticancer radiotherapy available to the clinician today (6). Since its cloning in 1996 NIS has been identified and characterized as a novel promising target gene for the treatment of extrathyroidal tumors following selective NIS gene transfer into tumor cells that allows diagnostic and therapeutic application of radioiodine and alternative radionuclides, such as 188 Re and 211 At (6-9). We have proven the feasibility of extrathyroidal radioiodine therapy after induction of iodide uptake by ex vivo

We recently demonstrated tumor-selective iodide uptake and therapeutic efficacy of combined radiovirotherapy after systemic delivery of the theranostic sodium iodide symporter (NIS) gene using a dendrimer-coated adenovirus. To further improve shielding and targeting we physically coated replication-selective adenoviruses carrying the hNIS gene with a conjugate consisting of cationic poly(amidoamine) (PAMAM) dendrimer linked to the peptidic, epidermal growth factor receptor (EGFR)-specific ligand GE11. In vitro experiments demonstrated coxsackie-adenovirus receptor-independent but EGFR-specific transduction efficiency. Systemic injection of the uncoated adenovirus in a liver cancer xenograft mouse model led to high levels of NIS expression in the liver due to hepatic sequestration, which were significantly reduced after coating as demonstrated by 123I-scintigraphy. Reduction of adenovirus liver pooling resulted in decreased hepatotoxicity and increased transduction efficiency in peripheral xenograft tumors. 124I-PET-imaging confirmed EGFR-specificity by significantly lower tumoral radioiodine accumulation after pretreatment with the EGFR-specific antibody cetuximab. A significantly enhanced oncolytic effect was observed following systemic application of dendrimer-coated adenovirus that was further increased by additional treatment with a therapeutic dose of 131I. These results demonstrate restricted virus tropism and tumor-selective retargeting after systemic application of coated, EGFR-targeted adenoviruses therefore representing a promising strategy for improved systemic adenoviral NIS gene therapy.