Podcasts about Huh7

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535922v1?rss=1 Authors: Tang, V. T., Abbineni, P. S., da Veiga Leprevost, F., Basrur, V., Emmer, B. T., Nesvizhskii, A., Ginsburg, D. Abstract: Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles/tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the LMAN1 or SURF4 gene. We found that LMAN1 has limited clients in HuH7 cells whereas SURF4 traffics a broad range of cargoes. Analysis of putative SURF4 cargoes suggests that cargo recognition is governed by complex mechanisms rather than interaction with a universal binding motif. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.22.517223v1?rss=1 Authors: Calla, J., Mittal, N., LaMonte, G., Liffner, B., Godinez-Macias, K., Carolino, K., Walker, G. T., Zou, B. Y., Paytas, E., Guerra, L., Tong-Rios, C., Campo, B., Vinetz, J. M., Gamboa, D., Raffatellu, M., Absalon, S., Winzeler, E. Abstract: Changes in host cell morphology and transcription after apicomplexan parasite infection have long been noted, but there have been few studies of the functional consequences of host cell remodeling. Here we show, using time-dependent immunofluorescence microscopy of multiple human cell lines (HFF, HepG2, HC-04, Huh7.5.1 and primary human hepatocytes), infected with multiple Plasmodium species (Plasmodium berghei, P. falciparum and P. vivax (hypnozoites and schizonts)), and antibodies to multiple human proteins (HsNR4A3, HsMUC13, HsGOLGA8A, HsCGA, HsBiP, HsCXCL2), that human protein trafficking is extensively modified in Plasmodium infected cells. Using conventional as well as ultrastructure expansion immunofluorescence microscopy we show that newly-synthesized human proteins are trafficked to the parasitophorous vacuole instead of the infected-cell plasma membrane, nucleus or extracellular space. Universal redirection of human signaling proteins cells the parasitophorous vacuole may provide a mechanistic explanation for how apicomplexan parasites can block host cells response to infection. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Researchers warn: Pfizer vaccine may affect integrity of genomic DNA. "Our study shows that BNT162b2 can be reverse transcribed to DNA in liver cell Huh7, and this may give rise to the concern if BNT162b2-derived DNA may be integrated into the host genome and affect the integrity of genomic DNA, which may potentially mediate genotoxic side effects", according to study authors. Then there's the situation with Ukraine, NATO, USA & UK. There's the impending food and supplies crisis. Covid-19 and lockdowns. Which of these situations pose as existential threat more and what steps do we need to take now, if any? Without question, we are living in the most challenging time most people alive now have ever known. As we each try to understand this period, and how best to navigate our way, what's the issue that needs immediate attention? Covid-19 & aftermath, including concer raised by the new study that Pfizer/BioNTech vaccine in vitro study, presenting evidence of it's fast entry into the cells & subsequent reverse transcription into DNA. Or, the impending food shortage WW3? What is your opinion? Please share if you like. Need wellness tips and information? Visit

Chemotherapeutic treatment of hepatocellular carcinoma often leads to chemoresistance during therapy or upon relapse of tumors. For the development of better treatments, a better understanding of biochemical changes in the resistant tumors is needed. Therefore, especially in vivo models are very important tools to generate standardized cell-material, which can be examined by high throughput techniques. Thus, it should be possible to find new targets for therapy or even for diagnostic. This thesis focusses on the characterization of the in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft mouse model. First step of the work was the establishment of the xenograft mouse model. SCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. After 10 weeks of response to the therapy, the tumor growth relapsed and tissue grew with very fast doubling time again, despite of ongoing treatment. This aggressive manner of growth under therapy could be also observed in a re-implantation study where the reisolated CPA chemoresistant HUH-REISO tumors grew without a lag phase, indicating an endogenous imprinted component. To evaluate this, tumors were examined by immunohistochemistry, a functional blood-flow Hoechst dye assay, and qRT-PCR for ALDH-1, Notch-1, Notch-3, HES-1, Thy-1, Oct-4, Sox-2 and Nanog mRNA levels. Histochemical analysis of HUH-REISO tumors revealed significant changes in host vascularization of tumors and especially in expression of the tumor-derived human endothelial marker gene PECAM-1/CD31 in HUH-REISO in comparison to parental HUH-7 cells and in vivo passaged HUH-PAS cells (in vivo grown without chemotherapeutic CPA pressure). The pronounced network of host murine vascularization in parental HUH-7 tumors was completely substituted by a network of human and murine vessel-like structures in HUH-REISO tumors under therapy. In addition, cell lines of these tumors were analyzed in endothelial trans-differentiation studies on matrigel. In those studies with limited oxygen and metabolite diffusion, followed by a matrigel assay, only the chemoresistant HUH-REISO cells exhibited tube formation potential and expression of human endothelial markers ICAM-2 and PECAM-1/CD31. Such a trans-differentiation capacity requires a lineage of cells with pluripotent capacities like so called tumor stem cells. Indeed, I could show in a comparative study on stemness and plasticity markers that Thy-1, Oct-4, Sox-2 and Nanog were upregulated in resistant xenografts. Furthermore, under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression, which I could also show by qRT-PCR. Notch-1 raised in HUH-PAS under therapeutic pressure, meanwhile it was conversely regulated in comparison to Thy-1, Oct-4, Sox-2 and Nanog in HUH-REISO. In both groups Notch-3 was inducible by 2 times CPA treatment and fell back on base level after further four therapeutic cycles in HUH-REISO. To conclude all these finding: chemoresistance of HUH-REISO was not manifested under standard in vitro, but only under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of trans-differentiation to endothelial like cells in vitro and in vivo. These cells expressed typical endothelial surface marker and functionality. Although the mechanism behind chemoresistance of HUH-REISO and involvement of plasticity remains to be clarified, we hypothesize that the observed Notch regulations and upregulation of stemness genes in resistant xenografts are involved in the observed cell plasticity.

Micro-patterned surfaces are frequently used in high-throughput single-cell studies, as they allow one to image isolated cells in defined geometries. Commonly, cells are seeded in excess onto the entire chip, and non-adherent cells are removed from the unpatterned sectors by rinsing. Here, we report on the phenomenon of cellular self-organization, which allows for autonomous positioning of cells on micro-patterned surfaces over time. We prepared substrates with a regular lattice of protein-coated adhesion sites surrounded by PLL-g-PEG passivated areas, and studied the time course of cell ordering. After seeding, cells randomly migrate over the passivated surface until they find and permanently attach to adhesion sites. Efficient cellular self-organization was observed for three commonly used cell lines (HuH7, A549, and MDA-MB-436), with occupancy levels typically reaching 40-60% after 3-5 h. The time required for sorting was found to increase with increasing distance between adhesion sites, and is well described by the time-to-capture in a random-search model. Our approach thus paves the way for automated filling of cell arrays, enabling high-throughput single-cell analysis of cell samples without losses.

Cancer is a leading cause of death worldwide. Although several common treatment options exist, there is an urgent need for improved tumor therapeutics. Therefore, we evaluated three innovative anticancer approaches in vivo. These were newly synthesized siRNA polyplexes, novel myxobacterial anticancer compounds, and innovative polymeric melphalan formulations. We evaluated the in vivo characteristics of polymeric bound siRNA. We could demonstrate that the polymer FolA-PEG24-K(Stp4-C)2 is an efficient carrier for targeting siRNA to the folate receptor expressing tumor tissue of mice. Moreover, the siRNA was able to enter tumor cells and led to specific gene silencing. After systemic injection, the polyplexes did neither cause any toxic side effects nor accumulate in any healthy organ. With only 6 nm average diameter, polyplexes were very small, resulting in fast removal from blood circulation by renal clearance. Addition of larger PEG spacers to the initial polyplex led to an increased polyplex size. As a result, the renal clearance was decreased, and polyplex distribution in the body was optimized. These results show that the in vivo hurdles of siRNA delivery can be overcome by binding siRNA to the precise and multifunctional polymers. Natural compounds have broad therapeutic effects, and are basis for the production of various anticancer drugs. Myxobacterial products often target cell structures which are rarely targeted by other metabolites, and they exert novel modes of action. Therefore, they are of particular importance for the development of new anticancer drugs. In this thesis, we evaluated three novel myxobacterial compounds in vivo. Within our experimental settings, we could demonstrate for the first time that archazolid, pretubulysin and chondramide can impair the migration and colonization of breast cancer cells in mice. Concerning their impact on tumor growth reduction, the compounds have promising characteristics, but the experimental setups need further improvement. Cytostatic drugs, like melphalan play an important role in cancer therapy, but bear problems, such as a low therapeutic efficacy and strong toxic side effects. Immobilization of chemotherapeutics on polymers is an interesting option to reduce their toxicity and enhance their efficacy, mainly by passive tumor targeting. We evaluated three innovative polymer-melphalan formulations in cell viability and proliferation assays. The covalent conjugate of poly(oxyethylene H-phosphonate) with melphalan was additionally investigated in vivo. This polymeric immobilization of melphalan led to an improved therapeutic effect compared to the pure cytostatic drug, as the growth and regrowth of HuH7 tumors could be hampered effectively. In conclusion, this thesis deals with the in vivo evaluation of innovative cancer therapeutics, which were successfully investigated in murine tumor models. The results with the experimental agents, based on siRNA, polymers or biogenic drugs, are encouraging starting points for further anticancer research.

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.

Background: Chemotherapeutic treatment of hepatocellular carcinoma often leads to chemoresistance during therapy or upon relapse of tumors. For the development of better treatments a better understanding of biochemical changes in the resistant tumors is needed. In this study, we focus on the characterization of in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft model. Methods: SCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. Tumors were evaluated by immunohistochemistry, a functional blood-flow Hoechst dye assay, and qRT-PCR for ALDH-1, Notch-1, Notch-3, HES-1, Thy-1, Oct-4, Sox-2 and Nanog mRNA levels. Cell lines of these tumors were analyzed by qRT-PCR and in endothelial transdifferentiation studies on matrigel. Results: HUH-REISO cells, although slightly more sensitive against activated CPA in vitro than parental HUH-7 cells, fully retained their in vivo CPA chemoresistance upon xenografting into SCID mice. Histochemical analysis of HUH-REISO tumors in comparison to parental HUH-7 cells and passaged HUH-PAS cells (in vivo passaged without chemotherapeutic pressure) revealed significant changes in host vascularization of tumors and especially in expression of the tumor-derived human endothelial marker gene PECAM-1/CD31 in HUH-REISO. In transdifferentiation studies with limited oxygen and metabolite diffusion, followed by a matrigel assay, only the chemoresistant HUH-REISO cells exhibited tube formation potential and expression of human endothelial markers ICAM-2 and PECAM-1/CD31. A comparative study on stemness and plasticity markers revealed upregulation of Thy-1, Oct-4, Sox-2 and Nanog in resistant xenografts. Under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression. Conclusions: Chemoresistance of HUH-REISO was not manifested under standard in vitro but under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of transdifferentiation to endothelial like cells in vitro and in vivo. These cells expressed typical endothelial surface marker and functionality. Although the mechanism behind chemoresistance of HUH-REISO and involvement of plasticity remains to be clarified, we hypothesize that the observed Notch regulations and upregulation of stemness genes in resistant xenografts are involved in the observed cell plasticity.

In order to deliver drugs to diseased cells nanoparticles featuring controlled drug release are developed. Controlled release is of particular importance for the delivery of toxic anti-cancer drugs that should not get in contact with healthy tissue. To evaluate the effectivity and controlled drug release ability of nanoparticles in the target cell, live-cell imaging by highly-sensitive fluorescence microscopy is a powerful method. It allows direct real-time observation of nanoparticle uptake into the target cell, intracellular trafficking and drug release. With this knowledge, existing nanoparticles can be evaluated, improved and more effective nanoparticles can be designed. The goal of this work was to study the internalization efficiency, successful drug loading, pore sealing and controlled drug release from colloidal mesoporous silica (CMS) nanoparticles. The entire work was performed in close collaboration with the group of Prof. Thomas Bein (LMU Munich), where the nanoparticles were synthesized. To deliver drugs into a cell, the extracellular membrane has to be crossed. Therefore, in the first part of this work, the internalization efficiency of PEG-shielded CMS nanoparticles into living HeLa cells was examined by a quenching assay. The internalization time scales varied considerably from cell to cell. However, about 67% of PEG-shielded CMS nanoparticles were internalized by the cells within one hour. The time scale is found to be in the range of other nanoparticles (polyplexes, magnetic lipoplexes) that exhibit non-specific uptake. Besides internalization efficiency, successful drug loading and pore sealing are important parameters for drug delivery. To study this, CMS nanoparticles were loaded with the anti-cancer drug colchicine and sealed by a supported lipid bilayer using a solvent exchange method (additional collaboration with the group of Prof. Joachim Rädler, LMU). Spinning disk confocal live-cell imaging revealed that the nanoparticles were taken up into HuH7 cells by endocytosis. As colchicine is known to exhibit toxicity towards microtubules, the microtubule network of the cells was destroyed within 2 h of incubation with the colchicine-loaded lipid bilayer-coated CMS nanoparticles. Although successful drug delivery was shown, it is necessary to develop controlled local release strategies. To achieve controlled drug release, CMS nanoparticles for redox-driven disulfide cleavage were synthesized. The particles contain the ATTO633-labeled amino acid cysteine bound via a disulfide linker to the inner volume. For reduction of the disulfide bond and release of cysteine, the CMS nanoparticles need to get into contact with the cytoplasmic reducing milieu of the target cell. We showed that nanoparticles were taken up by HuH7 cells via endocytosis, but endosomal escape seems to be a bottleneck for this approach. Incubation of the cells with a photosensitizer (TPPS2a) and photoactivation led to endosomal escape and successful release of the drug. In addition, we showed that linkage of ATTO633 at high concentration in the pores of silica nanoparticles results in quenching of the ATTO633 fluorescence. Release of dye from the pores promotes a strong dequenching effect providing an intense fluorescence signal with excellent signal-to-noise ratio for single-particle imaging. With this approach, we were able to control the time of photoactivation and thus the time of endosomal rupture. However, the photosensitizer showed a high toxicity to the cell, due to its presence in the entire cellular membrane. To reduce cell toxicity induced by the photosensitizer and to achieve spatial control on the endosomal escape, the photosensitizer protoporphyrin IX (PpIX) was covalently surface-linked to the CMS nanoparticles and used as an on-board photosensitizer (additional collaboration with the groups of Prof. Joachim Rädler and Prof. Heinrich Leonhardt, both LMU). The nanoparticles were loaded with model drugs and equipped with a supported lipid bilayer as a removable encapsulation. Upon photoactivation, successful drug delivery was observed. The mode of action is proposed as a two step cascade, where the supported lipid bilayer is disintegrated by singlet oxygen in a first step and the endosomal membrane ruptures enabling drug release in a second step. With this system, stimuli-responsive and controlled, localized endosomal escape and drug release is achieved. Taken together, the data presented in this thesis show that real-time fluorescence imaging of CMS nanoparticles on a single-cell level is a powerful method to investigate in great detail the processes associated with drug delivery. Barriers in the internalization and drug delivery are detected and can be bypassed via new nanoparticle designs. These insights are of great importance for improvements in the design of existing and the synthesis of new drug delivery systems.

Host-Guest chemistry based on mesoporous silica materials has attracted increasing attention in the past two decades. Potential applications for these functionalized materials are in the fields of controlled drug delivery, catalysis, separation or encapsulation of functional biomolecules. The present work is focused on the synthesis of nanosized, mesoporous drug delivery devices, which are able to release a preloaded drug as a result of a certain trigger action, e.g. during the endocytosis in a cancer cell. For this purpose, several different synthesis strategies had to be developed in order to incorporate the different required functional groups within one mesoporous silica nanoparticle. A spatial separation of two different functionalities was achieved by the development of a sequential co-condensation approach. With this approach, core-shell bifunctionalized colloidal mesoporous silica could be synthesized. The obtained particles are an important prerequisite for other systems presented in this work. The applicability of the copper-(I)-catalyzed Huisgen reaction (click reaction) as mild synthetic tool for the immobilization of biomolecules in the channels of mesoporous silica was investigated. In this joint project between our group and the research group of Prof. Ernst Wagner (LMU), it was shown that a sensitive enzyme can be immobilized with this strategy in the pores of SBA-15. It was demonstrated that the recoverability and long-term stability of the active enzyme benefits from the encapsulation in the host. The well-known strong biotin-avidin interaction was used for the construction of a protease-responsive cap system for controlling the release from colloidal mesoporous silica. Fluorescein was released from the nanoparticles as a model compound for small drug molecules. In order to monitor the release, a custom-made two-compartment fluorescence cuvette was designed. Thermoresponsive opening through protein denaturation was demonstrated for temperatures higher than 90 °C. A programmable opening temperature for this concept became possible by using DNA-linkers between the silica surface and the avidin cap. It was demonstrated that the length of the double-stranded DNA controls the opening temperature of the avidin cap. This work was carried out as a join project between our group, the research group of Prof. Thomas Carell (LMU) and the baseclick GmbH. Redox-responsive drug delivery was investigated in living cells. In this context, the release of disulfide-linked, dye-labeled cystein from the core of colloidal mesoporous silica was monitored by confocal fluorescence microscopy at a single cell level, in collaboration with the research group of Prof. Christoph Bräuchle. It was shown by photoinduced endosomal rupture that the endosomal escape is a bottleneck in redox-based drug delivery. This concept was extended through the synthesis of photosensitizer-functionalized, PEGylated colloidal mesoporous silica. It was demonstrated that particle-loaded endosomes collapse under irradiation with 405 nm light and release the particles into the cytosol. In another joint project between the groups of Prof. Bein, Prof. Bräuchle, Prof. Rädler and Prof. Leonhardt together with Dr. Ulrich Rothbauer (all LMU), the novel photosensitizer-functionalized porous nanoparticles were used as carriers for the delivery of small GFP-binding antibodies from Camelidae sp. into GFP-tubulin expressing HuH7 cancer cells. Additionally, the particles were encapsulated by a supported lipid bilayer. The attached photosensitizer was shown to play a key role in the delivery mechanism. Light-irradiation was used to destroy both surrounding membranes (supported lipid bilayer and endosomal membrane). Finally, the pH-responsive release of the membrane-intercalating peptide mellitin from a mesoporous SBA-15 host was demonstrated. This was possible through the use of pH-sensitive acetal linkers. The release of the peptide was shown by the lysis of mouse erythrocytes. This work was carried out in collaboration with the group of Prof. Ernst Wagner. To summarize, mesoporous silica materials were functionalized with different biomolecules in order to generate novel materials for potential applications in drug delivery or other controlled release applications. The newly developed concepts provide a basis for future work on mesoporous silica as a powerful and versatile drug delivery platform.

Gene therapy is a research area where nucleic acids are transferred into cells to treat neoplastic, metabolic and hereditary diseases. Delivery of genetic material into living organisms can be achieved with viral or non-viral vectors. Viral gene carriers are very efficient but present some major disadvantages due to their pathogenicity and immunogenicity. Nonviral carriers are based on synthetic molecules binding and condensing nucleic acids into small, virus-like particles. The aim of this thesis was to study the biodistribution and tumor targeting properties of non-viral gene vectors based on polyethylenimine (PEI) after systemic injection into mice. The gene vectors were labeled with fluorescent dyes emitting in the near infrared (NIR), which allowed studying their bio-distribution in living animal over time. Owing to its amine groups PEI has a high positive charge density that enables electrostatic interactions with negatively charged nucleic acids and their efficient compaction into nucleic acid-PEI complexes, called polyplexes. The net positive surface charge of these polyplexes permits interactions with negatively charged cell surface molecules, thus leading to their internalization into the cell. To avoid unspecific interactions with blood components and nontarget tissues after intravenous application, polyplexes were shielded with the hydrophilic molecule polyethylene glycol (PEG). PEI-based gene carrier systems were tested on two subcutaneously implanted tumor types: Human Hepatocellular Carcinoma (HUH7) and Murine Neuroblastoma (N2a). HUH7 cells express epidermal growth factor (EGF) receptors, while N2a cells express transferrin (Tf) receptors on their surfaces. To enable targeting of the polyplexes to the tumor cells, polyplexes were generated containing the ligands EGF and Tf for targeting of HUH7 cells and N2a cells respectively. The targeted polyplexes were then intravenously injected into immunodeficient, athymic nu/nu mice in which HUH7 or N2a tumor cells had been previously set under their skin. To monitor the biodistribution of polyplexes throughout the mouse organism and to evaluate their gene delivery capability into the neoplastic cells, polyplexes were labeled with fluorescent dyes (Alexa 750, NIR 797) or near infrared emitting quantum dots (QD), whose fluorescent expression signal was detected and analyzed with a device for imaging in vivo. All fluorescent molecules and quantum dots were biocompatible and non-toxic. They emitted light in the near infrared area of the spectrum, thus avoiding overlapping phenomena with autofluorescent biomolecules or absorption of light by hemoglobin. With all dyes used for polyplex labeling a fluorescent signal could be observed in organs like liver and lung being clearly distinguishable from background fluorescence. Among the fluorescent molecules tested, quantum dots were identified being the most suitable method for in vivo studies, showing the highest signal/noise ratios. PEG-shielding led to best tumor targeting efficiency when administering EGF or Tf-targeted polypelxes in mice bearing HUH7 and N2a tumors respectively. A clear fluorescent signal specific for tumor tissue was detected; the imaging software used allowed quantitative analysis of this signal. For this reason this system is now available for further experimental applications.

The advantages of non-viral gene transfer systems are safety and low immunogenicity, therefore they are well suited for use as vectors in gene therapy. The main disadvantage, namely their low gene-transfer-efficiency, can be improved through the development of systemic gene transfer systems using targeted vectors with high specificity and gene transfer efficiency. The intravenous application of PEI22lin/DNS complexes leads to a high gene expression in the lung, but with high toxicity. This observation can be explained by the positive surface charge of the DNS complexes and the uncomplexed free PEI, which leads to aggregation of erythrocytes. DNS complexes can be isolated from free uncomplexed PEI by gel filtration. The systemic application of gel filtrated PEI22lin complexes to non-tumor bearing mice resulted in reduced toxicity however there was a decreased in gene expression compared to non-filtrated complexes. The same experiment was performed on tumor bearing mice and again reduced toxicity was observed and interestingly slightly higher gene expression found in the tumor compared to the non-filtrated complexes. Shielding the positive surface charge of the PEI22lin complexes by transferrin led to increased gene expression in the tumor with reduced expression in the lung and other organs. The improved tumor targeted gene expression was associated with reduced systemic toxicity. Tumor targeted gene expression appears to be dependent on the tumor model as this observation was only found in neuro2A neuroblastoma tumor model in A/J mice and not in B16F10 melanoma tumor models of C57BL/6 mice and CT26 colon carcinoma tumor models of BALB/c mice. To enhance the intracellular efficiency of the vectors, the endosomolytic active peptide melittin was incorporated into the transferrin targeted complexes. This led to a further increase in gene expression in the Neuro2A tumor models in A/J mice. For the local gene transfer, electroporation proved to be an easy to handle method to obtain a high gene expression in tissue. The non-invasive kaliper electrode was suitable for gene transfer to both muscle and tumor. The applied voltage showed to be the most important parameter in expression. The use of electroporation for intratumoral transfer of the therapeutic gene encoding for the cytokine TNF was unsuccessful. However, systemic application of the TNF-α gene in transferrin targeted complexes in combination with the intraperitoneal application of the chemotherapeutic Doxil® showed a clear synergistic effect. A significant delay in the tumor growth and in some cases a complete regression of the tumor was observed. The enzyme cytochrome P450 metabolizes the non toxic prodrug cyclophosphamide (CPA) into the cytotoxic drug. Electroporation of the cytochrome P450 gene into the tumor lead to its localized protein expression. When followed by the intraperitoneal application of CPA, a significant delay in the tumor growth of the human hepatocellular carcinoma Huh7 was observed in SCID mice. When applied to the Neuro2A tumor model in A/J mice, this application scheme showed a complete tumor regression in two animals. Furthermore the systemic application of the P450 gene in transferrin targeted complexes containing melittin in combination with CPA led to a strong delay in the tumor growth. In summary, this work describes a new anti-cancer strategy using the combination of chemotherapeutics and non-viral gene delivery resulting in a synergistic therapeutic effect in vivo. This promising strategy will be more effective with the improvement of non-viral gene delivery systems which have better targeted gene expression with lower toxicity.