Imaging and targeted therapy of pancreatic ductal adenocarcinoma using the theranostic sodium iodide symporter (NIS) gene

The theranostic sodium iodide symporter (NIS) gene allows detailed molecular imaging of transgene expression and application of therapeutic radionuclides. As a crucial step towards clinical application, we investigated tumor specificity and transfection efficiency of epidermal growth factor receptor (EGFR)-targeted polyplexes as systemic NIS gene delivery vehicles in an advanced genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) that closely reflects human disease. PDAC was induced in mice by pancreas-specific activation of constitutively active Kras(G12D) and deletion of Trp53. We used tumor-targeted polyplexes (LPEIPEG-GE11/NIS) based on linear polyethylenimine, shielded by polyethylene glycol and coupled with the EGFR-specific peptide ligand GE11, to target a NIS-expressing plasmid to high EGFR-expressing PDAC. In vitro iodide uptake studies in cell explants from murine EGFR-positive and EGFR-ablated PDAC lesions demonstrated high transfection efficiency and EGFR-specificity of LPEI-PEG-GE11/NIS. In vivo I-123 gamma camera imaging and three-dimensional high-resolution I-124 PET showed significant tumor-specific accumulation of radioiodide after systemic LPEI-PEG-GE11/NIS injection. Administration of I-131 in LPEI-PEG-GE11/NIS-treated mice resulted in significantly reduced tumor growth compared to controls as determined by magnetic resonance imaging, though survival was not significantly prolonged. This study opens the exciting prospect of NIS-mediated radionuclide imaging and therapy of PDAC after systemic non-viral NIS gene delivery

Biodegradable poly (lactic acid-co-glycolic acid) scaffolds as carriers for geneticallymodified fibroblasts

Recent advances in gene delivery into cells allow improved therapeutic effects in gene therapy trials. To increase the bioavailability of applied cells, it is of great interest that transfected cells remain at the application site and systemic spread is minimized. In this study, we tested clinically used biodegradable poly(lactic acid-co-glycolic acid) (PLGA) scaffolds (Vicryl & Ethisorb) as transient carriers for genetically modified cells. To this aim, we used human fibroblasts and examined attachment and proliferation of untransfected cells on the scaffolds in vitro, as well as the mechanical properties of the scaffolds at four time points (1, 3, 6 and 9 days) of cultivation. Furthermore, the adherence of cells transfected with green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF165) and also VEGF165 protein secretion were investigated. Our results show that human fibroblasts adhere on both types of PLGA scaffolds. However, proliferation and transgene expression capacity were higher on Ethisorb scaffolds most probably due to a different architecture of the scaffold. Additionally, cultivation of the cells on the scaffolds did not alter their biomechanical properties. The results of this investigation could be potentially exploited in therapeutic regiments with areal delivery of transiently transfected cells and may open the way for a variety of applications of cell-based gene therapy, tissue engineering and regenerative medicine

EGFR-Targeted Adenovirus Dendrimer Coating for Improved Systemic Delivery of the Theranostic NIS Gene

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

Determination of mechanical properties for Vicryl and Ethisorb scaffolds with and without seeded cells.

<p>The mechanical properties of Vicryl (A) and Ethisorb (B) scaffolds were measured by using a uniaxial test system. Meshes were clamped along their long side with an initial length L₀ of 20 mm. Maximum force values for the two types of scaffolds (both with and without Hs27 cells) were measured in a single experiment at day (d) 1, 3, 6 and 9 and presented graphically (C). All error bars attached to the mean values represent the SEM of five technical replicates (n = 5; two-way ANOVA comparing maximum force values of Vicryl and Ethisorb scaffolds for each time point: ****p<0.00005).</p

Analysis of scaffold fibers and Hs27 cell morphology by scanning electron microscopy.

<p>Hs27 fibroblasts are grown on Vicryl and Ethisorb scaffolds and visualized at day 1, day 3 day6 and day 9 in culture by means of scanning electron microscopy (Scale bars in the upon two rows in A and B are 100μm, in the lowest row are 25μm)</p

Visualisation of Hs27 cell growth on Vicryl and Ethisorb scaffolds.

<p>During the co-cultivation period of 9 days, Hs27 fibroblast cells on Vicryl (A) and Ethisorb (B) scaffolds were investigated at four different time points by means of light microscopy with 100-fold magnification. In the upper row of each panel, the scaffolds containing no cells (control) are presented. The fibroblasts were visualized with Giemsa staining and can be seen in the images of the bottom row of each panel as small blue dots.</p