Novel promoter/transactivator configurations for macrolide- and streptogramin-responsive transgene expression in mammalian cells

Background

The recently developed heterologous macrolide‐ (E.REX system) and streptogramin‐ (PIP system) responsive gene regulation systems show significant differences in their regulation performance in diverse cell lines.

Methods

In order to provide optimal regulation modalities for a wide variety of mammalian cell lines, we have performed a detailed analysis of E.REX and PIP systems modified in (i) the transactivation domains of the antibiotic‐dependent transactivators, (ii) the type of minimal promoter used, and (iii) the spacing between the operator module and the minimal promoter.

Results

These novel E.REX and PIP regulation components showed not only dramatically improved regulation performance in some cell types, but also enabled their use in cell lines which had previously been inaccessible to regulated transgene expression.

Conclusions

Due to their modular set‐up the novel E.REX and PIP regulation systems presented here are most versatile and ready for future upgrades using different cell‐specific key regulation components. Copyright © 2002 John Wiley & Sons, Ltd.

     

Development and characterization of a minimal inducible packaging cell line for simian immunodeficiency virus-based lentiviral vectors

Background

Lentiviral vectors allow gene transfer into non‐dividing cells. Further development of these vector systems requires stable packaging cell lines that enable adequate safety testing.

Methods

To generate a packaging cell line for vectors based on simian immunodeficiency virus (SIV), expression plasmids were constructed that contain the codon‐optimized gag‐pol gene of SIV and the gene for the G protein of vesicular stomatitis virus (VSV‐G) under the control of an ponasterone‐inducible promoter. Stable cell lines expressing these packaging constructs were established and characterized.

Results

The RT activity and vector titers of cell clones stably transfected with the inducible gag‐pol expession plasmid could be induced by ponasterone by more than a factor of 1000. One of these clones was subsequently transfected with the ponasterone‐inducible VSV‐G expression plasmid to generate packaging cells. Clones of the packaging cells were screened for vector production by infection with an SIV vector and subsequent induction by ponasterone. In the supernatant of selected ponasterone‐induced producer clones vector titers of more than 1×10⁵ transducing units/ml were obtained. Producer cell clones were stable for at least five months, as tested by vector production.

Conclusions

The packaging cells described should be suitable for most preclinical applications of SIV‐based vectors. By avoiding regions of high homology between the vector and the packaging constructs, the design of the SIV packaging cell line should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the SIV vector constructs that can be packaged. Copyright © 2002 John Wiley & Sons, Ltd.

     

Evaluation of a porcine model for pulmonary gene transfer using a novel synthetic vector

Background

The pig lung, given its gross anatomical, histological and physiological similarities to the human lung, may be useful as a large animal model, in addition to rodents, in which to assess the potential of vectors for pulmonary airway gene transfer. The aim of this study was to assess the utility of the pig lung as a model of gene transfer to the human lung with a synthetic vector system.

Methods

The LID vector system consists of a complex of lipofectin (L), integrin‐binding peptide (I) and plasmid DNA (D). LID complexes containing a β‐galactosidase reporter gene under a CMV promoter or a control plasmid at1 mg/3 ml PBS, or 3 ml buffer, was administered to the right lower lobe ofthe pig lung through a bronchoscope. Pigs were culled at 48 h and lung sections prepared for immunohistochemical and histological analysis. Bronchoalveolar lavage fluid was collected and analysed for TNF‐α by ELISA.

Results

Immunohistochemical staining for the β‐galactosidase reporter gene indicated high efficiency of gene transfer by the LID vector to pig bronchial epithelium with 46% of large bronchi staining positively. There was no evidence for vector‐specific inflammation assessed by leukocytosis and cytokine production.

Conclusions

This study demonstrates the use of the pig for studies of gene transfer in the lung and confirms in a second species the potential of the LID vector for gene therapy of pulmonary diseases such as cystic fibrosis. Copyright © 2002 John Wiley & Sons, Ltd.

     

Polybrene and interleukin-4: two opposing factors for retroviral transduction of bone-marrow-derived dendritic cells

Background

Gene transfer using retroviral transduction offers the advantage of long‐term transgene expression in developing strategies that use dendritic cells (DCs) for immunotherapy. The goal of this study was to infect DCs in an immature state in order to take advantage of their proliferating and tolerogenic potential.

Methods

Immature DCs were generated from murine bone marrow (BM) using either GM‐CSF alone or GM‐CSF plus IL‐4. The cells were transduced directly with retroviral supernatants or by co‐culture with the GP + E‐86 retroviral packaging cell line in the presence of two different cationic polymers: polybrene and protamine sulfate. Phenotypic and functional characterization of the transduced cells were then performed.

Results

Our results show a low efficiency of retroviral infection of DCs in the presence of polybrene. This cationic polymer was found to be directly cytotoxic to murine DCs and thus favored the growth of contaminating macrophages. This effect was not observed using protamine sulfate. Furthermore, stimulation by IL‐4 early in the culture increased DC differentiation, proliferation and transduction. However, we found that DCs generated in GM‐CSF plus IL‐4 presented a more mature phenotype with an enhanced allogeneic stimulating activity. Finally, we showed that DCs themselves down‐regulated transgene expression in the co‐cultured packaging cell line in a promoter‐dependent manner.

Conclusions

We have defined optimal conditions to generate and transduce murine BM‐derived DCs. This included: the use of protamine sulfate during exposure to retroviral infectious supernatant and the addition of IL‐4 at an early stage of the culture. Nevertheless, this cytokine also induced DC maturation. These findings have potential implications in experimental gene therapy. Copyright © 2002 John Wiley & Sons, Ltd.

     

Polyethyleneimine-based immunopolyplex for targeted gene transfer in human lymphoma cell lines

Background

Specific and efficient delivery of genes into targeted cells is a priority objective in non‐viral gene therapy. Polyethyleneimine‐based polyplexes have been reported to be good non‐viral transfection reagents. However, polyplex‐mediated DNA delivery occurs through a non‐specific mechanism. This article reports the construction of an immunopolyplex, a targeted non‐viral vector based on a polyplex backbone, and its application in gene transfer over human lymphoma cell lines.

Methods

Targeting elements (biotin‐labeled antibodies), which should recognize a specific element of the target cell membrane and promote nucleic acid entry into the cell, were attached to the polyplex backbone through a bridge protein (streptavidin). Immunopolyplex transfection activity was studied in several hematological cell lines [Jurkat (CD3+/CD19?), Granta 519 (CD3?/ CD19+), and J.RT3‐T3.5 (CD3?/CD19?)] using the EGFP gene as a reporter gene and anti‐CD3 and anti‐CD19 antibodies as targeting elements. Transfection activity was evaluated via green fluorescence per cell and the percentage of positive cells determined by flow cytometry.

Results

A significant selectivity of gene delivery was observed, since the anti‐CD3 immunopolyplex worked only in Jurkat cells while the anti‐CD19 immunopolyplex worked only in the Granta cell line. Moreover, transfection of a CD3+/CD3? cell mixture with anti‐CD3 immunopolyplexes showed up to 16‐fold more transfection in CD3+ than in CD3? cells. Several non‐specific transfection reagents showed poor or no transfection activity.

Conclusion

It is concluded that immunopolyplex is a good non‐viral vector for specific and selective nucleic acid delivery. Immunopolyplex design allows easy replacement of the targeting element (antibody) – the streptavidin–polyplex backbone remaining intact – thereby conferring high versatility. Copyright © 2002 John Wiley & Sons, Ltd.

     

High-level gene transfer to the cornea using electroporation

Background

Methods for gene transfer to the cornea that yield high‐level expression without inflammation or trauma are currently lacking. Because electroporation has proven effective for gene transfer in other tissues in terms of expression levels and safety, this study quantitatively evaluated its use in the cornea.

Methods

To evaluate the use of electroporation in the mouse cornea, plasmids expressing either luciferase or green fluorescent protein were injected intracorneally or subconjunctivally and square‐wave electric pulses were immediately applied to the eyes. Gene expression was quantified at later times and trauma and inflammation were monitored visually and by measuring interleukin‐6 (IL‐6) production.

Results

The application of electric pulses to eyes injected with plasmid resulted in nanogram levels of gene product expression. At an optimal field strength of 200 V/cm, no trauma, corneal edema or inflammation was observed. However, at higher field strengths, corneal damage was detected. Compared with injection of DNA alone, up to 1000‐fold more gene product was produced using electroporation. Expression was detected as early as 6 h post‐electroporation, remained high for 3 days, and decreased by 7 days. Gene expression was detected over the entire surface of the cornea in both epithelial and stromal layers.

Conclusions

These results demonstrate that electroporation is an excellent method for delivering genes to multiple cell layers within the mouse cornea and that it results in extremely high levels of gene expression with little, if any, inflammatory response or tissue damage, making this a very useful technique for corneal gene transfer. Copyright © 2001 John Wiley & Sons, Ltd.

     

The use of low-molecular-weight PEIs as gene carriers in the monkey fibroblastoma and rabbit smooth muscle cell cultures

Background

Polyethylenimines (PEIs) and cationic polymers have been used successfully in gene delivery. In earlier reports, only large PEIs (MW>10 000) have shown significant transfection efficiency. In the present study, the roles of small PEIs (MW 700 and 2000) were studied as additional compounds to see if they can improve gene delivery with cationic liposomes.

Methods

The TKBPVlacZ expression plasmid was transfected in the CV1‐P (monkey fibroblastoma) and SMC (rabbit smooth muscle) cell lines using various combinations of PEIs (MW 700, 2000, and 25 000) and Dosper liposomes. The transfection efficiency was determined with the fluorometric ONPG (o‐nitrophenol‐β‐D ‐galactopyranoside) assay and histochemical X‐gal staining. The toxicity of the transfection reagents was estimated by the MTT [3‐(4,5‐dimethylthiazolyl‐2)‐2,5‐diphenyl tetrazolium bromide] assay.

Results

Transfection of TKBPVlacZ plasmid by the small PEIs (MW 700 and 2000) combined with Dosper liposomes was associated with high expression of the lacZ reporter gene in the CV1‐P and SMC cell lines. The transfection efficiencies of the low‐molecular‐weight PEI/liposome combinations were several fold higher than those of PEIs or liposomes alone. PEI/liposome combinations had no toxicity on the cell lines tested.

Conclusions

The low‐molecular‐weight PEIs could be used successfully for gene delivery when combined with the cationic liposomes, resulting in a synergistic increase of the transfection efficiency in both cell lines studied. Copyright © 2002 John Wiley & Sons, Ltd.

     

Highly efficient gene transfer system using a laminin–DNA–apatite composite layer

Background

We have recently developed a safe and efficient gene transfer system using a laminin–DNA–apatite composite layer. The objectives of the present study were to fully characterize and optimize the laminin–DNA–apatite composite layer in relation to the efficiency of gene transfer and to demonstrate the feasibility of the composite layer in the induction of cell differentiation.

Methods

The laminin–DNA–apatite composite layer was prepared under various conditions. The efficiency of gene transfer on the resulting composite layer was evaluated using luciferase and ß‐galactosidase gene expression assay systems. A laminin–DNA–apatite composite layer, prepared under the optimized condition using a plasmid including cDNA of nerve growth factor (NGF), was then applied to the neuron‐like differentiation of PC12 cells.

Results

The laminin content of the laminin–DNA–apatite composite layer was found to be a dominant factor improving the efficiency of gene transfer rather than the DNA content. The cell adhesion property of laminin in the composite layer should be responsible for the improvement in efficiency of gene transfer because the immobilization of albumin without the cell adhesion property in a DNA–apatite composite layer had no effect on the efficiency of gene transfer. A laminin–DNA–apatite composite layer, prepared under the optimized condition using a plasmid including cDNA of NGF, successfully induced the neuron‐like differentiation of PC12 cells.

Conclusions

The present gene transfer system, with the potential to control cell differentiation and having features of safety and relatively high and controllable efficiency, would be a useful tool for tissue engineering applications and the production of transfection microarrays. Copyright © 2010 John Wiley & Sons, Ltd.

     

High efficiency transfection of porcine vascular cells in vitro with a synthetic vector system

Background

Gene therapy strategies for the treatment of vascular disease such as the prevention of post‐angioplasty restenosis require efficient, non‐toxic transfection of vascular cells. In vitro studies in these cells contribute to vector development for in vivo use and for the evaluation of genes with therapeutic potential. The aim of this project was to evaluate a novel synthetic vector consisting of a liposome (L), an integrin targeting peptide (I), and plasmid DNA (D), which combine to form the LID vector complex.

Methods

Cultures of porcine smooth muscle cells and endothelial cells were established and then transfected with the LID vector, using the reporter genes luciferase and green fluorescent protein and the metalloprotease inhibitor TIMP‐1.

Results

The LID vector system transfected primary porcine vascular smooth muscle cells and porcine aortic endothelial cells with efficiency levels of 40% and 35%, respectively. By increasing the relative DNA concentration four‐fold, incubation periods as short as 30 min achieved the same levels of luciferase transgene expression as 4 h incubations at lower DNA concentrations. The transfection did not affect cell viability as measured by their proliferative potential. Serum levels of up to 20% in the transfection medium had no adverse affect on the efficiency of transfer and gene expression in either cell type. Transfections with the cDNA for TIMP‐1 produced protein levels that peaked at 130 ng/ml per 24 h and persisted for 14 days at 10 ng/ml per 24 h.

Conclusion

This novel vector system has potential for studies involving gene transfer to cardiovascular cells in vitro and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Lack of specificity of cell-surface protease targeting of a cytotoxic hyperfusogenic gibbon ape leukaemia virus envelope glycoprotein

Background

In a strategy termed “Protease Targeting”, retroviral vectors carrying an EGF infectivity‐blocking domain fused to the N‐terminus of the envelope SU via a MMP (matrix metalloproteinase)‐cleavable linker were successfully used to target gene delivery to EGF receptor‐(EGF‐R‐)positive tumour cells over‐expressing MMPs. In the current study, we aimed to investigate whether this strategy could be applied to (a) limit the cytotoxic activity of a hyperfusogenic GALV therapeutic gene, and (b) enhance the immune‐stimulatory properties of GALV via local, MMP‐mediated release human granulocyte‐macrophage colony stimulating factor (GM‐CSF).

Methods

We generated GALV envelope expression constructs displaying EGF or GM‐CSF blocking ligands at the N‐terminus of GALV envelope SU via a non‐cleavable, Factor Xa protease or MMP‐cleavable linker and investigated their cytotoxicity on MMP‐positive and negative cell lines.

Results

The unmodified hyperfusogenic GALV envelope was cytotoxic to all cell lines tested. The non‐cleavable linker GALV envelope constructs caused no cytotoxicity, demonstrating efficient inhibition by the displayed domains. Moderate activation of fusion of the protease‐cleavable linker constructs was observed in all cell lines, regardless of their level of MMP expression and of the specificity of the linker. High levels of the ‘blocking domain’ were detected in the cell supernatants due to dissociation of the surface unit (SU) from the transmembrane (TM) component of the GALV envelope glycoprotein TM.

Conclusions

Unlike protease targeting in the context of retroviral vectors, protease activation of the cytotoxicity of GALV envelope by cleavage of a fusion blocking ligand at the cell surface does not appear to be specifically mediated by cell‐surface MMPs. In addition, shedding of the SU‐fusion protein from the TM limits the general applicability of this strategy for cancer gene therapy. Specificity of cell‐cell fusion mediated by GALV envelope cannot be manipulated in the same fashion as virus‐cell fusion. Copyright © 2002 John Wiley & Sons, Ltd.

     

Cationic lipids derived from glycine betaine promote efficient and non-toxic gene transfection in cultured hepatocytes

Background

The low efficiency and toxicity of transfection in a primary culture of hepatocytes using cationic lipids remains a limiting step to the study of gene function and the setting up of non‐viral gene therapy.

Methods

A novel class of cationic lipids (GBs) derived from natural glycine betaine compounds covalently linked to acyl chains by enzymatically hydrolysable peptide and ester bonds, a structure designed to reduce cytotoxicity, was used to improve transfection efficiency in a primary culture of rat hepatocytes. The relationship between lipid structure, lipoplex formulation and transfection efficiency was studied using six GBs (12‐14‐16, 22‐24‐26) varying in their spacer and acyl chains.

Results

GB12, characterized by short [(CH₂)₁₀] acyl chains and spacer, allowed plasmid uptake in all cells and reporter gene expression in up to 40% of hepatocytes with a low cytotoxicity, a much higher efficiency compared with transfections using other reagents including Fugene6^? and Lipofectin^?. We also showed that numerous cells accumulated high amounts of plasmids demonstrating that GB12 promoted a very efficient DNA transfer through plasma membrane leading to an increase in nuclear plasmid translocation, allowing a much higher gene expression. Moreover, GB12‐transfected hepatocytes survived to injection in normal livers and were found to express the LacZ reporter gene.

Conclusions

The non‐toxic GB12 formulation is a powerful vehicle for plasmid delivery in cultured hepatocytes with relevance in liver gene therapy. Copyright © 2002 John Wiley & Sons, Ltd.

     

Syngeneic fibroblasts transfected with a plasmid encoding interleukin-4 as non-viral vectors for anti-inflammatory gene therapy in collagen-induced arthritis

Background

No effective long‐term treatment is available for rheumatoid arthritis. Recent advances in gene therapy and cell therapy have demonstrated efficiency in collagen‐induced arthritis (CIA). Interleukin‐4 (IL‐4) is already known to be efficient in CIA in systemic injection or administered by gene therapy. This study was designed to evaluate the effect of a non‐viral gene therapy of CIA, involving injection of syngeneic fibroblasts transfected with a plasmid encoding for IL‐4.

Methods

Immortalised fibroblasts from DBA/1 mice (DBA/1/0 cells) were transfected with a plasmid expressing IL‐4 cDNA (DBA/1/IL‐4 cells). Xenogeneic fibroblasts from Chinese hamster ovary (CHO) transfected with a plasmid expressing IL‐4 cDNA (CHO/IL‐4) were studied also. The cells were engrafted in mice developing CIA by subcutaneous injection of 3 × 10⁶ DBA/1/0 or DBA/1/IL‐4 or CHO/IL‐4 cells.

Results

Injection of DBA/1/IL‐4 cells, on days 10 and 25 after immunisation, was associated with a significant and lasting improvement in the clinical and histological evidence of joint inflammation and destruction as compared with DBA/1/0 and CHO/IL‐4 cells. DBA/1/IL‐4 cell treatment decreased also the production of IgG2a antibody to CII and the proliferation of CIIB‐specific nodal T cells. Later treatments (engraftments on days 23 and 35 after immunisation) exerted also an anti‐inflammatory effect, as evaluated on clinical and histological signs of CIA.

Conclusions

Taken together, these findings indicate that systemic administration of syngeneic cells transfected with an anti‐inflammatory cytokine gene, namely IL‐4, with a non‐viral method is effective in CIA and may attenuate the cytokine imbalance seen in this disease. Copyright © 2002 John Wiley & Sons, Ltd.

     

Receptor-mediated delivery of an antisense gene to human brain cancer cells

Background

The goal of this work was the development of a gene targeting technology that will enable the delivery of therapeutic genes to brain cancer cells in vivo following intravenous administration. High‐grade brain gliomas overexpress the epidermal growth factor receptor (EGFR) and EGFR antisense gene therapy could reduce the growth of EGFR‐dependent gliomas.

Methods

A human EGFR antisense gene driven by the SV40 promoter in a non‐viral plasmid carrying elements that facilitate extra‐chromosomal replication was packaged in the interior of 85 nm pegylated immunoliposomes (PILs). The PILs were targeted to U87 human glioma cells with the 83‐14 murine monoclonal antibody (MAb) to the human insulin receptor (HIR).

Results

Confocal fluorescent microscopy demonstrated that the unconjugated HIR MAb is rapidly internalized by the glioma cells. Endocytosis followed by entry into the nucleus was also demonstrated for the HIR MAb conjugated PILs carrying fluorescein‐labeled plasmid DNA. The PILs delivered exogenous genes to virtually all cells in culture, based on β‐galactosidase histochemistry. The targeting of a luciferase gene to the U87 cells with the PILs resulted in luciferase levels in excess of 150 pg/mg protein after 72 h of incubation. The level of luciferase gene expression in the U87 cells achieved with the PIL gene targeting system was comparable to that with lipofectamine. Targeting the EGFR antisense gene to U87 glioma cells with the PILs resulted in more than 70% reduction in [³H]thymidine incorporation into the cells; this was paralleled by a 79% reduction in the level of immunoreactive EGFR.

Conclusion

The present work describes the targeting of an EGFR antisense gene to human brain cancer cells, which results in a 70–80% inhibition in cancer cell growth. PILs provide a new approach to gene targeting that is effective in vivo following intravenous administration without viral vectors. Copyright © 2002 John Wiley & Sons, Ltd.