Generation of a tumor- and tissue-specific episomal non-viral vector system

Background: A key issue for safe and reproducible gene therapy approaches is the autologous and tissue-specific expression of transgenes. Tissue-specific expression in vivo is either achieved by transfer vectors that deliver the gene of interest into a distinct cell type or by use of tissue-specific expression cassettes. Here we present the generation of non-viral, episomally replicating vectors that are able to replicate in a tissue specific manner thus allowing tissue specific transgene expression in combination with episomal replication. The episomal replication of the prototype vector pEPI-1 and its derivatives depends exclusively on a transcription unit starting from a constitutively active promoter extending into the scaffold/matrix attachment region (S/MAR). Results: Here, we exchanged the constitutive promoter in the pEPI derivative pEPito by the tumor specific alpha fetoprotein (AFP) or the muscle specific smooth muscle 22 (SM22) promoter leading to specific transgene expression in AFP positive human hepatocellular carcinoma (HUH7) and in a SM22 positive cell line, respectively. The incorporation of the hCMV enhancer element into the expression cassette further boosted the expression levels with both promoters. Tissue specific-replication could be exemplary proven for the smooth muscle protein 22 (SM22) promoter in vitro. With the AFP promoter-driven pEPito vector hepatocellular carcinoma-specific expression could be achieved in vivo after systemic vector application together with polyethylenimine as transfection enhancer. Conclusions: In this study we present an episomal plasmid system designed for tissue specific transgene expression and replication. The human AFP-promoter in combination with the hCMV enhancer element was demonstrated to be a valuable tissue-specific promoter for targeting hepatocellular carcinomas with non-viral gene delivery system, and tissue specific replication could be shown in vitro with the muscle specific SM22 promoter. In combination with appropriate delivery systems, the tissue specific pEPito vector system will allow higher tissue-specificity with less undesired side effects and is suitable for long term transgene expression in vivo within gene therapeutical approaches

In vivo imaging enables high resolution preclinical trials on patients' leukemia cells growing in mice.

Xenograft mouse models represent helpful tools for preclinical studies on human tumors. For modeling the complexity of the human disease, primary tumor cells are by far superior to established cell lines. As qualified exemplary model, patients' acute lymphoblastic leukemia cells reliably engraft in mice inducing orthotopic disseminated leukemia closely resembling the disease in men. Unfortunately, disease monitoring of acute lymphoblastic leukemia in mice is hampered by lack of a suitable readout parameter

EGF Receptor-Targeted Synthetic Double-Stranded RNA Eliminates Glioblastoma, Breast Cancer, and Adenocarcinoma Tumors in Mice

BACKGROUND: Glioblastoma multiforme (GBM) is the most lethal form of brain cancer. With the available treatments, survival does not exceed 12–14 mo from the time of diagnosis. We describe a novel strategy to selectively induce the death of glioblastoma cells and other cancer cells that over-express the EGF receptor. Using a non-viral delivery vector that homes to the EGF receptor, we target synthetic anti-proliferative dsRNA (polyinosine-cytosine [poly IC]), a strong activator of apoptosis, selectively to cancer cells. METHODS AND FINDINGS: Poly IC was delivered by means of a non-viral vector: 25kDa polyethylenimine-polyethyleneglycol-EGF (PEI(25)-PEG-EGF). EGFR-targeted poly IC induced rapid apoptosis in the target cells in vitro and in vivo. Expression of several cytokines and “bystander killing” of untransfected tumor cells was detected in vitro and in vivo. Intra-tumoral delivery of the EGFR-targeted poly IC induced the complete regression of pre-established intracranial tumors in nude mice, with no obvious adverse toxic effects on normal brain tissue. A year after treatment completion the treated mice remain cancer-free and healthy. Similarly, non-viral delivery of poly IC completely eliminated pre-established breast cancer and adenocarcinoma xenografts derived from EGFR over-expressing cancer cell lines, suggesting that the strategy is applicable to other EGFR-over-expressing tumors. CONCLUSION: The strategy described has yielded an effective treatment of EGFR over-expressing GBM in an animal model. If this strategy is translated successfully to the clinical setting, it may actually offer help to GBM patients. Moreover the elimination of two additional EGFR over-expressing cancers in vivo suggests that in principle this strategy can be applied to treat other tumors that over-express EGFR

Live in vivo imaging of Egr-1 promoter activity during neonatal development, liver regeneration and wound healing

Background: The zinc finger transcription factor Egr-1 (Early growth response 1) is central to several growth factors and represents an important activator of target genes not only involved in physiological processes like embryogenesis and neonatal development, but also in a variety of pathophysiological processes, for example atherosclerosis or cancer. Current options to investigate its transcription and activation in vivo are end-point measurements that do not provide insights into dynamic changes in the living organism. Results: We developed a transgenic mouse (Egr-1-luc) in which the luciferase reporter gene is under the control of the murine Egr-1 promoter providing a versatile tool to study the time course of Egr-1 activation in vivo. In neonatal mice, bioluminescence imaging revealed a high Egr-1 promoter activity reaching basal levels three weeks after birth with activity at snout, ears and paws. Using a model of partial hepatectomy we could show that Egr-1 promoter activity and Egr-1 mRNA levels were increased in the regenerating liver. In a model of wound healing, we demonstrated that Egr-1 promoter activity was upregulated at the site of injury. Conclusion: Taken together, we have developed a transgenic mouse model that allows real time in vivo imaging of the Egr-1 promoter activity. The ability to monitor and quantify Egr-1 activity in the living organism may facilitate a better understanding of Egr-1 function in vivo. Additional File 1: BLI of adult Egr-1-luc mice with opened body cavity. Transgenic Egr-1-luc mice (one month old) received 6 mg luciferin in 100 μl PBS by intraperitoneal injection. Ten minutes thereafter the animal was killed by cervical dislocation, the body cavity opened immediately, skin from the ventral side partially removed and BLI measurement was carried out (10 min signal collection, setting 'high resolution'). A representative animal is shown with similar amplification setting as in Figure 2A

Novel PAMAM-PEG-Peptide Conjugates for siRNA Delivery Targeted to the Transferrin and Epidermal Growth Factor Receptors

The transferrin (TfR) and epidermal growth factor receptors (EGFR) are known to be overexpressed on the surface of a wide variety of tumor cells. Therefore, the peptides B6 (TfR specific) and GE11 (targeted to the EGFR) were linked to the PAMAM (polyamidoamine) structure via a polyethylenglycol (PEG) 2 kDa chain with the aim of improving the silencing capacity of the PAMAM-based dendriplexes. The complexes showed an excellent binding capacity to the siRNA with a maximal condensation at nitrogen/phosphate (N/P) 2. The nanoparticles formed exhibited hydrodynamic diameters below 200 nm. The zeta potential was always positive, despite the complexes containing the PEG chain in the structure showing a drop of the values due to the shielding effect. The gene silencing capacity was assayed in HeLa and LS174T cells stably transfected with the eGFPLuc cassette. The dendriplexes containing a specific anti luciferase siRNA, assayed at different N/P ratios, were able to mediate a mean decrease of the luciferase expression values of 14% for HeLa and 20% in LS174T cells, compared to an unspecific siRNA-control. (p < 0.05). In all the conditions assayed, dendriplexes resulted to be non-toxic and viability was always above 75%

pEPito: a significantly improved non-viral episomal expression vector for mammalian cells

<p>Abstract</p> <p>Background</p> <p>The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed <it>Cytomegalovirus </it>immediate early promoter (CMV-IEP) and directed into a 2000 bp long <it>matrix attachment region sequence </it>(MARS) derived from the human β-interferon gene. The original pEPI-1 vector contains two mammalian transcription units and a total of 305 CpG islands, which are located predominantly within the vector elements necessary for bacterial propagation and known to be counterproductive for persistent long-term transgene expression.</p> <p>Results</p> <p>Here, we report the development of a novel vector pEPito, which is derived from the pEPI-1 plasmid replicon but has considerably improved efficacy both <it>in vitro </it>and <it>in vivo</it>. The pEPito vector is significantly reduced in size, contains only one transcription unit and 60% less CpG motives in comparison to pEPI-1. It exhibits major advantages compared to the original pEPI-1 plasmid, including higher transgene expression levels and increased colony-forming efficiencies <it>in vitro</it>, as well as more persistent transgene expression profiles <it>in vivo</it>. The performance of pEPito-based vectors was further improved by replacing the CMV-IEP with the <it>human CMV enhancer/human elongation factor 1 alpha promoter </it>(hCMV/EF1P) element that is known to be less affected by epigenetic silencing events.</p> <p>Conclusions</p> <p>The novel vector pEPito can be considered suitable as an improved vector for biotechnological applications <it>in vitro </it>and for non-viral gene delivery <it>in vivo</it>.</p

Cadmium Telluride Quantum Dots as a Fluorescence Marker for Adipose Tissue Grafts

Plastic and reconstructive surgeons increasingly apply adipose tissue grafting in a clinical setting, although the anticipation of graft survival is insecure. There are only few tools for tracking transplanted fat grafts in vivo. Murine adipose tissue clusters were incubated with negatively charged, mercaptoproprionic acid-coated cadmium telluride quantumdots (QDs) emitting in the dark red or near infrared. The intracellular localization of QDs was studied by confocal laser scanning microscopy. As a result, the adipose tissue clusters showed a proportional increase in fluorescence with increasing concentrations (1, 10, 16, 30, 50 nM) of cadmium telluride QDs. Laser scanning microscopy demonstrated a membrane bound localization of QDs. Vacuoles and cell nuclei of adipocytes were spared by QDs. We conclude that QDs were for the first time proven intracellular in adult adipocytes and demonstrate a strong fluorescence signal. Therefore, they may play an essential role for in vivo tracking of fat grafts

Correlated Multimodal Imaging in Life Sciences:Expanding the Biomedical Horizon

International audienceThe frontiers of bioimaging are currently being pushed toward the integration and correlation of several modalities to tackle biomedical research questions holistically and across multiple scales. Correlated Multimodal Imaging (CMI) gathers information about exactly the same specimen with two or more complementary modalities that-in combination-create a composite and complementary view of the sample (including insights into structure, function, dynamics and molecular composition). CMI allows to describe biomedical processes within their overall spatio-temporal context and gain a mechanistic understanding of cells, tissues, diseases or organisms by untangling their molecular mechanisms within their native environment. The two best-established CMI implementations for small animals and model organisms are hardware-fused platforms in preclinical imaging (Hybrid Imaging) and Correlated Light and Electron Microscopy (CLEM) in biological imaging. Although the merits of Preclinical Hybrid Imaging (PHI) and CLEM are well-established, both approaches would benefit from standardization of protocols, ontologies and data handling, and the development of optimized and advanced implementations. Specifically, CMI pipelines that aim at bridging preclinical and biological imaging beyond CLEM and PHI are rare but bear great potential to substantially advance both bioimaging and biomedical research. CMI faces three mai

Amorphous Silica Particles Relevant in Food Industry Influence Cellular Growth and Associated Signaling Pathways in Human Gastric Carcinoma Cells

Nanostructured silica particles are commonly used in biomedical and biotechnical fields, as well as, in cosmetics and food industry. Thus, their environmental and health impacts are of great interest and effects after oral uptake are only rarely investigated. In the present study, the toxicological effects of commercially available nano-scaled silica with a nominal primary diameter of 12 nm were investigated on the human gastric carcinoma cell line GXF251L. Besides the analysis of cytotoxic and proliferative effects and the comparison with effects of particles with a nominal primary diameter of 200 nm, emphasis was also given to their influence on the cellular epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases (MAPK) signaling pathways—both of them deeply involved in the regulation of cellular processes like cell cycle progression, differentiation or proliferation. The investigated silica nanoparticles (NPs) were found to stimulate cell proliferation as measured by microscopy and the sulforhodamine B assay. In accordance, the nuclear level of the proliferation marker Ki-67 was enhanced in a concentration-dependent manner. At high particle concentrations also necrosis was induced. Finally, silica NPs affected the EGFR and MAPK pathways at various levels dependent on concentration and time. However, classical activation of the EGFR, to be reflected by enhanced levels of phosphorylation, could be excluded as major trigger of the proliferative stimulus. After 45 min of incubation the level of phosphorylated EGFR did not increase, whereas enhanced levels of total EGFR protein were observed. These results indicate interference with the complex homeostasis of the EGFR protein, whereby up to 24 h no impact on the transcription level was detected. In addition, downstream on the level of the MAP kinases ERK1/2 short term incubation appeared to affect total protein levels without clear increase in phosphorylation. Depending on the concentration range, enhanced levels of ERK1/2 phosphorylation were only observed after 24 h of incubation. Taken together, the present study demonstrates the potential of the tested silica particles to enhance the growth of gastric carcinoma cells. Although interference with the EGFR/MAPK cascade is observed, additional mechanisms are likely to be involved in the onset of the proliferative stimulus