Theranostic Gene Research Articles

UBE2C: A pan-cancer diagnostic and prognostic biomarker revealed through bioinformatics analysis.

The diverse and complex attributes of cancer have made it a daunting challenge to overcome globally and remains to endanger human life. Detection of critical cancer-related gene alterations in solid tumor samples better defines patient diagnosis and prognosis, and indicates what targeted therapies must be administered to improve cancer patients' outcome. To identify genes that have aberrant expression across different cancer types, differential expressed genes were detected within the TCGA datasets. Subsequently, the DEGs common to all pan cancers were determined. Furthermore, various methods were employed to gain genetic alterations, co-expression genes network and protein-protein interaction (PPI) network, pathway enrichment analysis of common genes. Finally, the gene regulatory network was constructed. Intersectional analysis identified UBE2C as a common DEG between all 28 types of studied cancers. Upregulated UBE2C expression was significantly correlated with OS and DFS of 10 and 9 types of cancer patients. Also, UBE2C can be a diagnostic factor in CESC, CHOL, GBM, and UCS with AUC = 100% and diagnose 19 cancer types with AUC ≥90%. A ceRNA network constructed including UBE2C, 41 TFs, 10 shared miRNAs, and 21 circRNAs and 128 lncRNAs. In summary, UBE2C can be a theranostic gene, which may serve as a reliable biomarker in diagnosing cancers, improving treatment responses and increasing the overall survival of cancer patients and can be a promising gene to be target by cancer drugs in the future.

The sodium iodide symporter (NIS) as theranostic gene: potential role in pre-clinical therapy of extra-thyroidal malignancies

The sodium iodide symporter (NIS) as theranostic gene: potential role in pre-clinical therapy of extra-thyroidal malignancies

Mesenchymal Stem Cell-mediated Image-guided Sodium Iodide Symporter (NIS) Gene Therapy Improves Survival of Glioblastoma-bearing Mice.

Mesenchymal stem cells (MSC) have emerged as cellular-based vehicles for the delivery of therapeutic genes in cancer therapy based on their inherent tumor-homing capability. As theranostic gene, the sodium iodide symporter (NIS) represents a successful target for noninvasive radionuclide-based imaging and therapy. In this study, we applied genetically engineered MSCs for tumor-targeted NIS gene transfer in experimental glioblastoma (GBM)-a tumor with an extremely poor prognosis. A syngeneic, immunocompetent GL261 GBM mouse model was established by subcutaneous and orthotopic implantation. Furthermore, a subcutaneous xenograft U87 model was used. Bone marrow-derived MSCs were stably transfected with a NIS-expressing plasmid driven by the constitutively active cytomegalovirus promoter (NIS-MSC). After multiple or single intravenous injection of NIS-MSCs, tumoral iodide uptake was monitored in vivo using 123I-scintigraphy or 124I-PET. Following validation of functional NIS expression, a therapy trial with 131I was performed on the basis of the most optimal application regime as seen by 124I-PET imaging in the orthotopic approach. A robust tumoral NIS-specific radionuclide accumulation was observed after NIS-MSC and radioiodide application by NIS-mediated in vivo imaging. NIS immunofluorescence staining of GBM and non-target tissues showed tumor-selective MSC homing along with NIS expression. Application of therapeutically effective 131I led to significantly delayed tumor growth and prolonged median survival after NIS-MSC treatment as compared with controls. A strong tumor-selective recruitment of systemically applied MSCs into GBM was found using NIS as reporter gene followed by successful therapeutic application of radioiodide demonstrating the potential use of NIS-based MSCs as therapy vehicles as a new GBM therapy approach.

Polymeric nanoparticles for dual-targeted theranostic gene delivery to hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) develops predominantly in the inflammatory environment of a cirrhotic liver caused by hepatitis, toxin exposure, or chronic liver disease. A targeted therapeutic approach is required to enable cancer killing without causing toxicity and liver failure. Poly(beta-amino-ester) (PBAE) nanoparticles (NPs) were used to deliver a completely CpG-free plasmid harboring mutant herpes simplex virus type 1 sr39 thymidine kinase (sr39) DNA to human HCC cells. Transfection with sr39 enables cancer cell killing with the prodrug ganciclovir and accumulation of 9-(4-18F-fluoro-3-hydroxymethylbutyl)guanine (18F-FHBG) for in vivo imaging. Targeting was achieved using a CpG-free human alpha fetoprotein (AFP) promoter (CpGf-AFP-sr39). Expression was restricted to AFP-producing HCC cells, enabling selective transfection of orthotopic HCC xenografts. CpGf-AFP-sr39 NP treatment resulted in 62% reduced tumor size, and therapeutic gene expression was detectable by positron emission tomography (PET). This systemic nanomedicine achieved tumor-specific delivery, therapy, and imaging, representing a promising platform for targeted treatment of HCC.

Development and Characterization of Ultrasound Activated Lipopolyplexes for Enhanced Transfection by Low Frequency Ultrasound in In Vitro Tumor Model.

This work focuses on the development of ultrasound contrast vesicles for ultrasound-mediated enhanced transfection of nucleic acids in the cancer cells and projects its application as a tool for diagnostic imaging. The ultrasound contrast vesicles are stable, anionic, nanoscaled vesicles with ultrasound contrast equivalent to the commercially available SonoVue. These anionic lipid vesicles establish electrostatic interaction with cationic polyplexes based on linear polyethylenimine (22kDa)forming lipopolyplexes with ultrasound contrast. The lipopolyplexes are characterized regarding shape, size, and zeta potential. When exposed to low frequency ultrasound, these carriers show elevated transfection efficiency and reduced cytotoxicity. The effect of post-transfection ultrasound on cellular uptake of lipopolyplexes is also evaluated. An analogous transfection is also observed in the tumor mimicking multicellular 3D spheroid culture of ovarian cancer cells. The emergence of tumor imaging and enhanced gene delivery by medical ultrasound, a noninvasive imaging modality, is considered paving the way for efficient theranostic gene therapy.

Dual-targeted NIS polyplexes-a theranostic strategy toward tumors with heterogeneous receptor expression.

Tumor heterogeneity, within and between tumors, may have severe implications for tumor therapy, especially for targeted gene therapy, where single-targeted approaches often result in limited efficacy and therapy resistance. Polymer-formulated nonviral vectors provide a potent delivery platform for cancer therapy. To improve applicability for future clinical use in a broad range of patients and cancer types, a dual-targeting approach was performed. Synthetic LPEI-PEG2kDa-based polymer backbones were coupled to two tumor-specific peptide ligands GE11 (EGFR-targeting) and cMBP (cMET-targeting). The dual-targeting approach was used to deliver the theranostic sodium iodide symporter (NIS) gene to hepatocellular cancer. NIS as auspicious theranostic gene allows noninvasive imaging of functional NIS gene expression and effective anticancer radioiodide therapy. Enhanced tumor-specific transduction efficiency of dual-targeted polyplexes compared to single-targeted polyplexes was demonstrated in vitro using tumor cell lines with different EGFR and cMET expression and in vivo by 124I-PET-imaging. Therapeutic efficacy of the bispecific concept was mirrored by significantly reduced tumor growth and perfusion, which was associated with prolonged animal survival. In conclusion, the dual-targeting approach highlights the benefits of a bifunctional strategy for a future clinical translation of the bioimaging-based NIS-mediated radiotherapy allowing efficient targeting of heterogeneic tumors with variable receptor expression levels.

A Novel Approach for Image-Guided 131I Therapy of Pancreatic Ductal Adenocarcinoma Using Mesenchymal Stem Cell-Mediated NIS Gene Delivery.

The sodium iodide symporter (SLC5A5/NIS) as theranostic gene would allow for non-invasive imaging of functional NIS expression and therapeutic radioiodine application. Genetically engineered mesenchymal stem cells (MSC), based on their tumor-homing abilities, show great promise as tumor-selective NIS gene delivery vehicles for non-thyroidal tumors. As a next step towards clinical application, tumor specificity and efficacy of MSCs were investigated in an advanced genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC). Syngeneic murine MSCs were stably transfected with a NIS-expressing plasmid driven by the CMV-promoter (NIS-MSC). In vivo 123I-scintigraphy and 124I-PET revealed significant perchlorate-sensitive NIS-mediated radioiodide accumulation in PDAC after systemic injection of NIS-MSCs. Active MSC recruitment into the tumor stroma was confirmed using NIS immunohistochemistry (IHC). A therapeutic strategy, consisting of three cycles of systemic MSC-mediated NIS delivery, followed by 131I application, resulted in a significant delay and reduction in tumor growth as compared to controls. Furthermore, IHC analysis of α-SMA and Ki67 revealed differences in the amount and behavior of activated fibroblasts in tumors of mice injected with NIS-MSCs as compared with saline-treated mice. Taken together, MSCs as NIS gene delivery vehicles in this advanced endogenous PDAC mouse model demonstrated high stromal targeting of NIS by selective recruitment of NIS-MSCs after systemic application resulting in an impressive 131I therapeutic effect. IMPLICATIONS: These data expand the prospect of MSC-mediated radioiodine imaging-guided therapy of pancreatic cancer using the sodium iodide symporter as a theranostic gene in a clinical setting.

Systemic tumor-targeted sodium iodide symporter (NIS) gene therapy of hepatocellular carcinoma mediated by B6 peptide polyplexes.

Nonviral polymer-based gene transfer represents an adaptable system for tumor-targeted gene therapy because various design strategies of shuttle systems, together with the mechanistic concept of active tumor targeting, lead to improved gene delivery vectors resulting in higher tumor specificity, efficacy and safety. Using the sodium iodide symporter (NIS) as a theranostic gene, nonviral gene delivery vehicles based on linear polyethylenimine (LPEI), polyethylene glycol (PEG) and coupled to the synthetic peptide B6 (LPEI-PEG-B6), which specifically binds to tumor cells, were investigated in a hepatocellular carcinoma xenograft model for tumor selectivity and transduction efficiency. In vitro incubation of three different tumor cell lines with LPEI-PEG-B6/NIS resulted in significant increase in iodide uptake activity compared to untargeted and empty vectors. After establishment of subcutaneous HuH7 tumors, NIS-conjugated nanoparticles were injected intravenously followed by analysis of radioiodide biodistribution using 123 I-scintigraphy showing significant perchlorate-sensitive iodide accumulation in tumors of LPEI-PEG-B6/NIS-treated mice (8.0±1.5% ID/g 123 I; biological half-life of 4h). After four cycles of repetitive polyplex/131 I applications, a significant delay of tumor growth was observed, which was associated with markedly improved survival in the therapy group. These results clearly demonstrate that systemic in vivo NIS gene transfer using nanoparticle vectors coupled to B6 tumor targeting ligand is capable of inducing tumor-specific radioiodide uptake. This promising gene therapy approach opens the exciting prospect of NIS-mediated radionuclide therapy in metastatic cancer, together with the possibility of combining several targeting ligands to enhance selective therapeutic efficacy in a broad field of cancer types with various receptor expression profiles.

Sequence-defined cMET/HGFR-targeted Polymers as Gene Delivery Vehicles for the Theranostic Sodium Iodide Symporter (NIS) Gene

The sodium iodide symporter (NIS) as well-characterized theranostic gene represents an outstanding tool to target different cancer types allowing noninvasive imaging of functional NIS expression and therapeutic radioiodide application. Based on its overexpression on the surface of most cancer types, the cMET/hepatocyte growth factor receptor serves as ideal target for tumor-selective gene delivery. Sequence-defined polymers as nonviral gene delivery vehicles comprising polyethylene glycol (PEG) and cationic (oligoethanoamino) amide cores coupled with a cMET-binding peptide (cMBP2) were complexed with NIS-DNA and tested for receptor-specificity, transduction efficiency, and therapeutic efficacy in hepatocellular cancer cells HuH7. In vitro iodide uptake studies demonstrated high transduction efficiency and cMET-specificity of NIS-encoding polyplexes (cMBP2-PEG-Stp/NIS) compared to polyplexes without targeting ligand (Ala-PEG-Stp/NIS) and without coding DNA (cMBP2-PEG-Stp/Antisense-NIS). Tumor recruitment and vector biodistribution were investigated in vivo in a subcutaneous xenograft mouse model showing high tumor-selective iodide accumulation in cMBP2-PEG-Stp/NIS-treated mice (6.6 ± 1.6% ID/g (123)I, biological half-life 3 hours) by (123)I-scintigraphy. Therapy studies with three cycles of polyplexes and (131)I application resulted in significant delay in tumor growth and prolonged survival. These data demonstrate the enormous potential of cMET-targeted sequence-defined polymers combined with the unique theranostic function of NIS allowing for optimized transfection efficiency while eliminating toxicity.

Sequence defined cMET/HGFR-targeted polymers as gene delivery vehicles for the theranostic sodium iodide symporter (NIS) gene

The sodium iodide symporter (NIS) as well-characterized theranostic gene represents an outstanding tool to target different cancer types allowing noninvasive imaging of functional NIS expression and therapeutic radioiodide application. Based on its overexpression on the surface of most cancer types, the cMET/hepatocyte growth factor receptor serves as ideal target for tumor-selective gene delivery. Sequence-defined polymers as nonviral gene delivery vehicles comprising polyethylene glycol (PEG) and cationic (oligoethanoamino) amide cores coupled with a cMET-binding peptide (cMBP2) were complexed with NIS-DNA and tested for receptor-specificity, transduction efficiency, and therapeutic efficacy in hepatocellular cancer cells HuH7. In vitro iodide uptake studies demonstrated high transduction efficiency and cMET-specificity of NIS-encoding polyplexes (cMBP2-PEG-Stp/NIS) compared to polyplexes without targeting ligand (Ala-PEG-Stp/NIS) and without coding DNA (cMBP2-PEG-Stp/Antisense-NIS). Tumor recruitment and vector biodistribution were investigated in vivo in a subcutaneous xenograft mouse model showing high tumor-selective iodide accumulation in cMBP2-PEG-Stp/NIS-treated mice (6.6 ± 1.6% ID/g 123 I, biological half-life 3 hours) by 123 I-scintigraphy. Therapy studies with three cycles of polyplexes and 131 I application resulted in significant delay in tumor growth and prolonged survival. These data demonstrate the enormous potential of cMET-targeted sequence-defined polymers combined with the unique theranostic function of NIS allowing for optimized transfection efficiency while eliminating toxicity.

338. Improving Virus-Free Modification of Endothelial Cells - Magnetically Targeted Manipulation

Regulation of angiogenesis is recognized as a crucial component in the treatment of multiple diseases, e.g. cardiovascular disorders, cancers, diabetes. At the same time, gene therapy is a promising tool for angiogenesis manipulation. Its wide clinical application is, however, limited by the lack of suitable theranostic gene delivery systems. To address this issue, in this study we intended to develop an efficient method for magnetically guided manipulation of endothelial cells. First, we defined optimal conditions for HUVEC transfection with microRNA (miR) using polyethyleneimine-magnetic nanoparticle based vector (PEI/MNP), pre-designed in our group. We found that miRNA can be efficiently delivered with PEI/MNP vector without affecting cell survival and functionality. Furthermore, the presence of magnetic component offers certain targeting and non-invasive MRI monitoring opportunities, which we investigated in vitro. Two different approaches were assessed: direct vector guidance and magnetic targeting of transfected cells. As a result, production of miR/PEI/MNP-modified magnetically responsive cells has been selected for further investigation as it was more efficient. We have demonstrated in vitro that up to 80% of transfected cells can be driven to the site of interest by magnetic field. In addition, properties of engineered cells were monitored up to 96 hours and proposed modification strategy has been proven safe. Further, transfected cells were tested in vitro in terms of MRI detection potential. We observed that 104 cells can be detected, when loaded with at least 0.2 pg iron/cell. This and higher intracellular iron concentrations were achieved at optimal transfection conditions defined previously. Moreover, we showed that miR/PEI/MNP-modified endothelial cells do maintain intercellular gap-junctional communication and potentially can exchange miR, serving thereby as its carrier. Taken together, obtained results are indicating that miR/PEI/MNP-modified cells are particularly promising as a multifunctional tool for magnetically guided angiogenesis regulation.View Large Image | Download PowerPoint Slide

A synthetic biology-based device prevents liver injury in mice

The liver performs a panoply of complex activities coordinating metabolic, immunologic and detoxification processes. Despite the liver's robustness and unique self-regeneration capacity, viral infection, autoimmune disorders, fatty liver disease, alcohol abuse and drug-induced hepatotoxicity contribute to the increasing prevalence of liver failure. Liver injuries impair the clearance of bile acids from the hepatic portal vein which leads to their spill over into the peripheral circulation where they activate the G-protein-coupled bile acid receptor TGR5 to initiate a variety of hepatoprotective processes. By functionally linking activation of ectopically expressed TGR5 to an artificial promoter controlling transcription of the hepatocyte growth factor (HGF), we created a closed-loop synthetic signalling network that coordinated liver injury-associated serum bile acid levels to expression of HGF in a self-sufficient, reversible and dose-dependent manner. After implantation of genetically engineered human cells inside auto-vascularizing, immunoprotective and clinically validated alginate-poly-(L-lysine)-alginate beads into mice, the liver-protection device detected pathologic serum bile acid levels and produced therapeutic HGF levels that protected the animals from acute drug-induced liver failure. Genetically engineered cells containing theranostic gene circuits that dynamically interface with host metabolism may provide novel opportunities for preventive, acute and chronic healthcare. Liver diseases leading to organ failure may go unnoticed as they do not trigger any symptoms or significant discomfort. We have designed a synthetic gene circuit that senses excessive bile acid levels associated with liver injuries and automatically produces a therapeutic protein in response. When integrated into mammalian cells and implanted into mice, the circuit detects the onset of liver injuries and coordinates the production of a protein pharmaceutical which prevents liver damage.

Mesenchymal stem cell-mediated, tumor stroma-targeted radioiodine therapy of metastatic colon cancer using the sodium iodide symporter as theranostic gene.

The tumor-homing property of mesenchymal stem cells (MSCs) allows targeted delivery of therapeutic genes into the tumor microenvironment. The application of sodium iodide symporter (NIS) as a theranostic gene allows noninvasive imaging of MSC biodistribution and transgene expression before therapeutic radioiodine application. We have previously shown that linking therapeutic transgene expression to induction of the chemokine CCL5/RANTES allows a more focused expression within primary tumors, as the adoptively transferred MSC develop carcinoma-associated fibroblast-like characteristics. Although RANTES/CCL5-NIS targeting has shown efficacy in the treatment of primary tumors, it was not clear if it would also be effective in controlling the growth of metastatic disease. To expand the potential range of tumor targets, we investigated the biodistribution and tumor recruitment of MSCs transfected with NIS under control of the RANTES/CCL5 promoter (RANTES-NIS-MSC) in a colon cancer liver metastasis mouse model established by intrasplenic injection of the human colon cancer cell line LS174t. RANTES-NIS-MSCs were injected intravenously, followed by (123)I scintigraphy, (124)I PET imaging, and (131)I therapy. Results show robust MSC recruitment with RANTES/CCL5-promoter activation within the stroma of liver metastases as evidenced by tumor-selective iodide accumulation, immunohistochemistry, and real-time polymerase chain reaction. Therapeutic application of (131)I in RANTES-NIS-MSC-treated mice resulted in a significant delay in tumor growth and improved overall survival. This novel gene therapy approach opens the prospect of NIS-mediated radionuclide therapy of metastatic cancer after MSC-mediated gene delivery.

Theranostic studies of human sodium iodide symporter imaging and therapy using 188Re: a human glioma study in mice.

ObjectiveTo investigate the role of 188Re in human sodium iodide symporter (hNIS) theranostic gene-mediated human glioma imaging and therapy in model mice.MethodsThe human glioma cell line U87 was transfected with recombinant lentivirus encoding the hNIS gene under the control of cytomegalovirus promoter (U87-hNIS). The uptake and efflux of 188Re were determined after incubating the cells with 188Re. 188Re uptake experiments in the presence of various concentrations of sodium perchlorate were carried out. In vitro cell killing tests with 188Re were performed. U87-hNIS mediated 188Re distribution, imaging and therapy in nude mice were also tested.ResultsU87-hNIS cell line was successfully established. The uptake of 188Re in U87-hNIS cells increased up to 26-fold compared to control cells, but was released rapidly with a half-life of approximately 4 minutes. Sodium perchlorate reduced hNIS-mediated 188Re uptake to levels of control cell lines. U87-hNIS cells were selectively killed following exposure to 188Re, with a survival of 21.4%, while control cells had a survival of 92.1%. Unlike in vitro studies, U87-hNIS tumor showed a markedly increased 188Re retention even 48 hours after 188Re injection. In the therapy study, there was a significant difference in tumor size between U87-hNIS mice (317±67 mm3) and control mice (861±153 mm3) treated with 188Re for 4 weeks (P<0.01).ConclusionThe results indicate that inserting the hNIS gene into U87 cells is sufficient to induce specific 188Re uptake, which has a cell killing effect both in vitro and in vivo. Moreover, our study, based on the function of hNIS as a theranostic gene allowing noninvasive imaging of hNIS expression by 188Re scintigraphy, provides detailed characterization of in vivo vector biodistribution and level, localization, essential prerequisites for precise planning and monitoring of clinical gene therapy that aims to individualize gene therapy concept.

Cationic polythiophenes as responsive DNA-binding polymers

A new water soluble cationic polythiophene derivative, poly(N,N,N-trimethyl-3-(2-(thiophen-3-yl)acetamido)propan-1-aminium iodide), was synthesized via two consecutive post-polymerization functionalizations of poly(methyl 2-(thiophen-3-yl)acetate). This conjugated polymer binds DNA at N/P = 5 and forms polyplexes at N/P = 10. Its potential use as a theranostic gene delivery vehicle is investigated here.

Systemic Image-Guided Liver Cancer Radiovirotherapy Using Dendrimer-Coated Adenovirus Encoding the Sodium Iodide Symporter as Theranostic Gene

Currently, major limitations for the clinical application of adenovirus-mediated gene therapy are high prevalence of neutralizing antibodies, widespread expression of the coxsackie-adenovirus receptor (CAR), and adenovirus sequestration by the liver. In the current study, we used the sodium iodide symporter (NIS) as a theranostic gene to investigate whether coating of adenovirus with synthetic dendrimers could be useful to overcome these hurdles in order to develop adenoviral vectors for combination of systemic oncolytic virotherapy and NIS-mediated radiotherapy. We coated replication-deficient (Ad5-CMV/NIS) (CMV is cytomegalovirus) and replication-selective (Ad5-E1/AFP-E3/NIS) adenovirus serotype 5 carrying the hNIS gene with poly(amidoamine) dendrimers generation 5 (PAMAM-G5) in order to investigate transduction efficacy and altered tropism of these coated virus particles by (123)I scintigraphy and to evaluate their therapeutic potential for systemic radiovirotherapy in a liver cancer xenograft mouse model. After dendrimer coating, Ad5-CMV/NIS demonstrated partial protection from neutralizing antibodies and enhanced transduction efficacy in CAR-negative cells in vitro. In vivo (123)I scintigraphy of nude mice revealed significantly reduced levels of hepatic transgene expression after intravenous injection of dendrimer-coated Ad5-CMV/NIS (dcAd5-CMV/NIS). Evasion from liver accumulation resulted in significantly reduced liver toxicity and increased transduction efficiency of dcAd5-CMV/NIS in hepatoma xenografts. After PAMAM-G5 coating of the replication-selective Ad5-E1/AFP-E3/NIS, a significantly enhanced oncolytic effect was observed after intravenous application (virotherapy) that was further increased by additional treatment with a therapeutic dose of (131)I (radiovirotherapy) and was associated with markedly improved survival. These results demonstrate efficient liver detargeting and tumor retargeting of adenoviral vectors after coating with synthetic dendrimers, thereby representing a promising innovative strategy for systemic NIS gene therapy. Moreover, our study-based on the function of NIS as a theranostic gene allowing the noninvasive imaging of NIS expression by (123)I scintigraphy-provides detailed characterization of in vivo vector biodistribution and localization, level, and duration of transgene expression, essential prerequisites for exact planning and monitoring of clinical gene therapy trials that aim to individualize the NIS gene therapy concept.