Low Transfection Rates Research Articles

Oncolytic adenouiruses in combination with chemtherapy in tumor therapy

In recent years oncolytic adenoviruses in combination with chemtherapy used in the treat-ment research of cancer attract more and more attention. The ability of tumor cells to develop chemotherapy drug resistance is a major limitation of standard anticaner drugs.Oncolytic adenovirus can overcome the disadvantage of traditional gene therapy involving a replication-deficient adenovirus that results in lower expression of the therapeutic gene with low transfection rate and no targeting effect,and also can improve the killing effect of tra-ditional chemotherapy. It is reasonable to suggest that oncolytic adenoviruses in combination with chemtherapy may be a powerful and effective protocol for cancer treatment in the clinic. Key words: Neoplasms; Adenoviridae; Genes; Drug therapy

Suspended-drop electroporation for high-throughput delivery of biomolecules into cells

We present a high-throughput method that enables efficient delivery of biomolecules into cells. The device consists of an array of 96 suspended electrode pairs, where small sample volumes are top-loaded, electroporated and bottom-ejected into 96-well plates. We demonstrate the use of this suspended-drop electroporation (SDE) device to effectively introduce fluorescent dextran, small interfering RNA (siRNA) or cDNA into primary neurons, differentiated neutrophils and other cell types with conventionally low transfection rates.

A dimerizable cationic lipid with potential for gene delivery

Despite the use of currently optimized lipofection conditions, including transfection in serum-depleted media, the efficiency of gene transfer is low and high transfection rates often induce cytotoxicity. A lipid formulation with transfection efficiency not inhibited by serum would provide an advance towards in vivo applications. We explored the ability of the cationic lipid SH-14 to dimerize upon DNA and form lipoplexes, and potentially release nucleic acids in the intracellular reducing milieu. We investigated the critical micelle-forming concentration of SH-14 and its intrinsic toxicity, size and Zeta potential measurements, the in vitro cytotoxicity of SH-14/plasmid DNA lipoplexes and their ability to transfect cells. Among all the charge ratios (CR, + /-) tested, lipoplexes at CR 10 with a mean diameter of 295 nm and a surface charge of + 20 mV, exhibited the best compromise between transfection efficiency and tolerability. SH-14 presented the same cytotoxicity level whether alone or complexed in lipoplexes. Lipofections carried out in serum-free medium shared a transfection efficiency, on average, of 40% and a cytotoxicity of 38%. An increase of 73% in transfection efficiency and 24% in cell viability were obtained, extending lipofection over 48 h in complete-medium. Moreover, when serum concentration was increased from 10% to 50%, a three-fold increase in plasmid dose led to more than 72% of cells being transfected with almost no sign of cytotoxicity. Overall, SH-14 presents good potential as a novel transfection reagent to be used in the presence of serum.

Rationale for the Selection of an Aerosol Delivery System for Gene Delivery

Genetic therapeutics show great promise toward the treatment of illnesses associated with the lungs; however, current methods of delivery such as jet and ultrasonic nebulization decrease the activity and effectiveness of these treatments. Extremely low transfection rates exhibited by non-complexed plasmid DNA in these nebulizers have been primarily attributed to poor translocation and loss of molecular integrity as a consequence of shear-induced degradation. Current research focusing on methods to increase transfection rates via the pulmonary delivery route has largely concentrated on the incorporation of carbon dioxide in the air stream to increase breath depth as well as the addition of cationic agents that condense DNA into compact, ordered complexes. The purpose of this study was to examine the impact of several classic as well as the latest atomization devices on the structure of non-complexed DNA. Various sizes of plasmid and cosmid DNA were processed through an electrostatic spray, ultrasonic nebulizer, vibrating mesh nebulizer, and jet nebulizer. Results varied dramatically based upon atomization device as well as DNA size. This may explain the inefficiency experienced by genetic therapeutics during pulmonary delivery. More importantly, this suggests that the selection of an atomization device should consider DNA size in order to achieve optimal gene delivery to the lungs.

Functional transfer of eukaryotic expression plasmids to mammalian cells by Listeria monocytogenes: a mechanistic approach

Cystic fibrosis (CF) is one of the most common monogenic disorders in the caucasian population. Gene therapy for CF is principally feasible and bacterial transfer systems might provide novel possibilities for therapy. However, transfection efficiencies are low and need to be improved. Thus, more detailed understanding of the DNA transfer mechanism is necessary to systematically eliminate these restrictions. Functional transfer of GFP-CFTR (cystic fibrosis transmembrane conductance regulator) to eukaryotic cells using attenuated Listeria monocytogenes mediated gene transfer (bacteriofection) was shown by fluorescent microscopy, flow cytometry, immunoblotting and whole cell patch clamping. The characteristics of plasmid transfer were studied by use of electron and fluorescence microscopy, flow cytometry and Southern blotting. Polymerase chain reaction (PCR) was used to screen the genome of bacteriofected cells for cotransfer of chromosomal bacterial DNA. Correct intracellular localization and functionality of the GFP-CFTR fusion protein after bacteriofection was shown. Efficient bacterial lysis and release of bacterial content was demonstrated using antibiotics to kill intracellular bacteria. Although only low transfection rates were observed, high numbers of transferred plasmids were detected in host cells under these conditions. However, they were associated with high molecular weight entities and not available to cytosolic transcription. Cotransfer of bacterial genomic DNA was observed in transfectants but occurred at low frequencies. In this work we demonstrate that low rates of bacteria-mediated transfection are not due to poor invasion of bacteria, insufficient bacterial lysis, or plasmid DNA degradation. Our data suggest that the transferred plasmid DNA is associated with higher macromolecular structures inhibiting nuclear transport and transgene transcription.

Eradication of p53-mutated head and neck squamous cell carcinoma (HNSCC) xenografts using nonviral p53 gene therapy and photochemical internalisation (PCI)

3155 Background: p53 gene therapy trials have been initiated using viral vectors in p53-mutated HNSCC. Alternatively, using nonviral vectors for gene therapy is limited because of their low transfection rate. Our in vitro results using PCI demonstrated that PCI enhances the transfection efficiency of polyethylenimine-mediated p53 gene transfer and justify in vivo evaluation. Methods: Tetraglucosylated polyethylenimine (PEIGlu4)-mediated gene transfer was performed alone or in conjunction with PCI, in mice bearing HNSCC xenograft. Using luciferase as reporter, we evaluate the influence of PCI on the transgene expression kinetics. Then, p53 gene transfer was performed by weekly injection of p53/PEIGlu4 complexes during five (Group A) or seven (Group B) consecutive weeks. Efficiency of the gene therapy was evaluated by following tumor growth and by analysis of P53, P21, mdm2, Bax and Bcl2 proteins expression. Results: Using PCI, the expression of luciferase was significantly (P < 0.01) enhanced. Enhancement was maximal (20-fold) at 48h and remained significant during 7 days after completion of the transfection protocol. Applying PCI to p53 gene transfer, tumor growth inhibition was observed in all transfected animals. One week after the last PCI-mediated p53 gene transfer, complete tumor regression was achieved in 4/6 mice (Group A) and in 6/6 mice (Group B). Three weeks after the transfection tumor regrowth was observed in 3/4 mice (Group A) and in 1/6 mice (Group B). Successful tumor cure was achieved in 1 mouse (Group A) and in 5/6 mice (Group B) that were still tumor-free 8 weeks after the last transfection. The use of PCI led to higher P53 protein expression and was correlated with subsequent induction of Bax, P21 and MDM2 proteins and with repression of Bcl2 protein. Conclusions: PCI enhances the in vivo efficiency of PEIGlu4-mediated p53 nonviral gene transfer and can be proposed as a potent alternative for p53 gene therapy in HNSCC. No significant financial relationships to disclose.

306. A Novel Thymidine Kinase in Enhanced Gap Junction Mediated Suicide Gene Therapy

Top of pageAbstract Patients with malignant gliomas have a poor prognosis due to failure of conventional therapy, and new treatment paradigms are urgently needed. The extensive heterogeneity of glioblastoma cells calls for a treatment strategy with multiple (cellular) targets. Suicide gene therapy for malignant gliomas has showed promising results in experimental animal models, but has failed in clinical trials due to low transfection rate, insufficient expression of the transgene in tumor cells, and low bystander killing effect. Gap junction communication (GJC) - allowing intercellular transport of small water soluble molecules - is generally low in malignant cells, leading to insufficient bystander effect. We have previously shown that treatment of glioma cells with the histone deacetylase inhibitor 4-phenyl butyrate (PB) leads to upregulation of the gap junction protein connexin 43 expression in both its phosphorylated and non-phosphorylated forms 1. This correlates with enhanced intercellular gap junction communication and increased bystander killing effect in a herpes simplex thymidine kinase (HSVtk)/ganciclovir suicide gene therapy in vitro model 2. A novel plant thymidine kinase with high affinity for the nucleoside analogue AZT was employed in a suicide gene therapy in vitro model. We show that the plant-TK/AZT system is superior to the HSV-TK/GCV system (app. 450- vs 55-fold time of activation) in the glioblastoma cell line U-87 MG. In addition, U-87 MG cells recombinantly expressing this thymidine kinase, showed a synergistic sensitivity to an AZT/PB combination therapy. This effect was shown to be partially gap junction mediated. Similar to glioblastoma cells, we show that neural stem cells (NSC) are subject to enhanced gap junction communication by treatment with PB. This was shown by enhanced fluorescent dye transfer between NSCs and glioblastoma cells. Furthermore, the bystander killing of tumor cells was investigated using plant kinase expressing NSCs co-cultured with glioblastoma cells in the presence of an AZT/PB combination compared to AZT alone. The presence of PB also resulted in an upregulation of connexin 43 in NSCs, as was shown for glioma cells. The successful cell killing in vitro using the plant-TK/AZT system prompted us to test this in vivo. An intracranial glioblastoma xenograft model in immuno-suppressed rats was developed. Plant-TK expressing U-87 MG glioma cell xenografts were established. The sensitivity of the tumor cells to AZT was assessed by intracranial infusion by use of an osmotic minipump. Drug concentrations of 40, 80, and 160 mM, with a flow of 5 ml/hour were used. 80 mM AZT was well tolerated by the rats for a 2 week period. The anti-tumor effect was assessed by histological and immunohistochemical techniques after sacrifice of animals and sectioning of the brain. To explore the NSC migration in rat brains carrying glioma xenografts, BrdU labeled NSCs or NSCs recombinantly expressing green fluorescent protein have been injected at some distance from the tumor site and their migrational capacity is being studied.

Lipid-mediated gene transfer of acidic fibroblast growth factor into human corneal endothelial cells

The aim of this study was to optimize non-viral gene transfer conditions and investigate the effect of fibroblast growth factor-1 (FGF-1) gene transfer on human corneal endothelial cell (HCEC) proliferation. Five non-viral vectors (Lipofectin™, DMRIE-C™, DAC-30, Effectene™, FuGene™6) were used to transfect HCEC with plasmids coding for enhanced green fluorescent protein (EGFP) and FGF-1. Transfection efficiency and toxicity ( n=6) were quantified and optimized using the EGFP construct by FACS-analysis. Using optimal conditions HCEC were transfected with the FGF-1 plasmid and cell proliferation as well as expression of FGF-1 were determined at days 4 and 7 by counting and western blotting, respectively. Lipofectin (17±2·02%) transfected HCEC more successfully than DMRIE-C (11±1·46%), Effectene (9±0·62%), FuGene (9±0·93%) and DAC-30 (7±0·59%). Toxicity of the lipids ranged from 2 to 4%. Optimal HCEC proliferation was achieved with DAC-30/FGF-1 ( P<0·05), whereas all other vectors did not result in significantly increased cell proliferation. However, all of the transfected cells produced FGF-1 in different amounts as indicated by western blotting. Efficient and almost non-toxic transfer of the FGF-1 gene into HCEC can be successfully achieved by lipid-based techniques. Using optimal conditions significantly increased cell proliferation was independent on gene transfer efficiency. This may indicate that even a low transfection rate is sufficient to produce a concentration of FGF-1 that will have a stimulatory effect on HCECs.

HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells

Malignant glioma patients have a dismal prognosis with an urgent need of new treatment modalities. Previously developed gene therapies for brain tumors showed promising results in experimental animal models, but failed in clinical trials due to low transfection rates and insufficient expression of the transgene in tumor cells, as well as low bystander killing effects. We have previously shown that the histone deacetylase inhibitor 4-phenylbutyrate (4-PB) enhances gap junction communication between glioma cells in culture. In this study, we demonstrate an activation of recombinant HSV-tk gene expression, and a dramatic enhancement of gap junction-mediated bystander killing effect by administration of the HSV-tk prodrug ganciclovir together with 4-PB. These findings that 4-PB potentiates “suicide gene” expression as well as enhances gap junctional communication and bystander killing of tumor cells justify further testing of this paradigm as an adjunct to suicide gene therapy of malignant gliomas.

A rapid and versatile method for harnessing scFv antibody fragments with various biological effector functions

A versatile expression vector is described for the rapid construction and evaluation of bispecific scFvs and scFv-based fusion proteins. An important feature of this vector is the presence of two multiple cloning sites (MCS) separated by an in frame linker sequence. The first MCS was specifically designed to contain unique SfiI and NotI restriction enzyme sites that can be used for directional and in frame insertion of scFvs (or potentially any molecule) selected from established phage-display systems. Using this new vector, a functional bs-(scFv) 2 (2C11-MOC31) was constructed for retargeted T-cell cytotoxicity towards EGP2 positive tumor cells. The vector was also used for grafting of a number of promising biological effector principles onto scFv MOC31, including the prodrug converting enzyme cytosine deaminase, the anti-angiogenic factor angiostatin, and the thrombogenic molecule tissue factor. We aimed at producing biologically active fusion proteins by directing them through the endoplasmic reticulum-based protein folding machinery of eukaryotic cells (COS-7) using a kappa light chain leader, thereby taking advantage of the associated quality control mechanisms that allow only fully folded and processed fusion proteins to be secreted into the medium. Supernatants derived from fusion protein transfected COS-7 cells, which were transiently transfected at low transfection rates, were directly assayed for the biological and/or targeting activity of the excreted fusion proteins without any prior purification steps. This procedure might help to identify those fusion proteins that have favourable characteristics like stability and biological activity in the presence of serum and at low protein concentrations. Targeted delivery of all effector principles was subsequently assessed in an in vitro model system. The method we devised is both rapid and versatile and can be useful to construct and identify series of new chimeric proteins with enhanced therapeutic potential in human cancer therapy.

Direct gene transfer into Actinidia deliciosa protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS-based method

Chloramphenicol acetyl transferase (CAT) gene was used as a reporter gene to assess the conditions for polyethylene glycol (PEG)-mediated transfection of kiwifruit protoplasts. The effect of plasmid concentration and the presence of carrier DNA were each assessed by analysing CAT activity in transfected protoplasts using thin-layer chromatography (TLC) autoradiographic detection of acetylated chloramphenicol. A gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) non-radioactive method was developed for monitoring CAT gene activity. This method provides a high speed of analysis (30 min) and precise means of detecting acetylated products at the nanomolar level, enabling quantification at very low transfection rates. Using this method we optimized plasmid and PEG concentration and also assessed the effect of heat shock on transfection. The best CAT activity was obtained using 30% polyethylene glycol 4000 and by submitting protoplasts to heat shock (45 degrees C, 5 min) prior to transfection.