RNAi screening identifies TAK1 as a potential target for the enhanced efficacy of topoisomerase inhibitors.

In an effort to develop strategies that improve the efficacy of existing anticancer agents, we have conducted a siRNA-based RNAi screen to identify genes that, when targeted by siRNA, improve the activity of the topoisomerase I (Top1) poison camptothecin (CPT). Screening was conducted using a set of siRNAs corresponding to over 400 apoptosisrelated genes in MDA-MB-231 breast cancer cells. During the course of these studies, we identified the silencing of MAP3K7 as a significant enhancer of CPT activity. Follow-up analysis of caspase activity and caspase-dependent phosphorylation of histone H2AX demonstrated that the silencing of MAP3K7 enhanced CPT-associated apoptosis. Silencing MAP3K7 also sensitized cells to additional compounds, including CPT clinical analogs. This activity was not restricted to MDA-MB-231 cells, as the silencing of MAP3K7 also sensitized the breast cancer cell line MDA-MB-468 and HCT-116 colon cancer cells. However, MAP3K7 silencing did not affect compound activity in the comparatively normal mammary epithelial cell line MCF10A, as well as some additional tumorigenic lines. MAP3K7 encodes the TAK1 kinase, an enzyme that is central to the regulation of many processes associated with the growth of cancer cells (e.g. NF- κB, JNK, and p38 signaling). An analysis of TAK1 signaling pathway members revealed that the silencing of TAB2 also sensitizes MDA-MB-231 and HCT-116 cells towards CPT. These findings may offer avenues towards lowering the effective doses of Top1 inhibitors in cancer cells and, in doing so, broaden their application.

Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes.

Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.

Gene therapy progress and prospects. Downregulating gene expression: the impact of RNA interference.

The control and maintenance of gene expression is critical for cell development and differentiation. Over the last 2 years, our understanding of the role of RNA as a regulator of gene expression has significantly increased. Small RNA molecules are key elements of a machinery that trigger chromosomal modifications, post-transcriptional gene silencing and protein translational blockade depending on the source, the RNA and the nature of the interaction with the target nucleic acid. Currently, the best characterized of this group of RNA-mediated gene regulation pathways is the post-transcriptional gene silencing mechanism known as RNA interference. RNAi is triggered by double-stranded RNA (dsRNA), which induces the formation of a ribonucleoprotein complex that mediates sequence-specific cleavage of the transcript cognate with the input dsRNA. RNAi has been adapted as a functional genomics tool and it has potential as a therapeutic approach. This review will summarize our current understanding of the RNAi mechanism and the various applications of RNAi-based technologies.

Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.

Short interfering RNAs (siRNAs) are double-stranded RNAs of approximately 21-25 nucleotides that have been shown to function as key intermediaries in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates. siRNAs have a characteristic structure, with 5'-phosphate/3'-hydroxyl ends and a 2-base 3' overhang on each strand of the duplex. In this study, we present data that synthetic siRNAs can induce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice. In each case, the interference by siRNAs was superior to the inhibition of gene expression mediated by single-stranded antisense oligonucleotides. The siRNAs seem to avoid the well documented nonspecific effects triggered by longer double-stranded RNAs in mammalian cells. These observations may open a path toward the use of siRNAs as a reverse genetic and therapeutic tool in mammalian cells.

dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference.

RNA interference (RNAi) is a form of post-transcriptional gene silencing that has been described in a number of plant, nematode, protozoan, and invertebrate species. RNAi is characterized by a number of features: induction by double stranded RNA (dsRNA), a high degree of specificity, remarkable potency and spread across cell boundaries, and a sustained down-regulation of the target gene. Previous studies of RNAi have examined this effect in whole organisms or in extracts thereof; we have now examined the induction of RNAi in tissue culture. A screen of mammalian cells from three different species showed no evidence for the specific down-regulation of gene expression by dsRNA. By contrast, RNAi was observed in Drosophila Schneider 2 (S2) cells. Green fluorescent protein (GFP) expression in S2 cells was inhibited in a dose-dependent manner by transfection of dsRNA corresponding to gfp when GFP was expressed either transiently or stably. This effect was structure- and sequence-specific in that: (1) little or no effect was seen when antisense (or sense) RNA was transfected; (2) an unrelated dsRNA did not reduce GFP expression; and (3) dsRNA corresponding to gfp had no effect on the expression of an unrelated target transgene. This invertebrate tissue culture model should allow facile assays for loss of function in a well-defined cellular system and facilitate further understanding of the mechanism of RNAi and the genes involved in this process.

Adeno-retroviral chimeric viruses as in vivo transducing agents.

Several hybrid viral gene transfer systems have been described that exploit the favorable features of the two parent viral species. We have developed a hybrid adeno-retroviral vector system to generate a retroviral vector in situ. The system consists of adenoviruses encoding MoMLV gag.pol (Axtet.gag.pol), the VSV-G viral envelope (Axtet.VSV-G), the retroviral vector LXSN expressing the neomycin phosphotransferase gene (AV-LXSN) and a transcriptional regulator to control expression of gag.pol and envelope (AV-rtTA). In vitro, biologically active retroviral vector preparations were generated following adeno-retroviral transduction of 9L rat glioma cells. In vivo the transcomplementing adeno-retroviruses were co-administered intratumorally into subcutaneous 9L glioma tumors in rats and human A375 melanoma xenografts in nude mice. In the 9L rat model, G418 cell cultures were only obtained when 9L cells were harvested from tumors injected with all four transcomplementing adeno-retroviruses. Molecular analysis of DNA extracted from 9L G418 populations derived both in vitro and in vivo showed appropriate integration of the LXSN proviral sequence. Tumor cells were harvested 1, 3 and 4 weeks after adeno-retrovirus administration to the human A375 xenografts. The percentage of G418 colonies recovered from tumors transduced with all of the transcomplementing adeno-retroviruses increased with time, whereas no increase was observed in tumors transduced with AV-LXSN alone. DNA extracted from G418 A375 cell populations showed the presence of integrated proviral sequences only in animals that received the full complement of adeno-retroviruses. These results demonstrate that adenoviral vectors expressing transcomplementing genes for retroviral proteins and retroviral vector RNAs can be used for in situ transduction of target cells.

The effects of jet nebulisation on cationic liposome-mediated gene transfer in vitro.

Nebulisation is currently the most acceptable and practical delivery system for repeated applications of gene therapy to the lower airways of cystic fibrosis (CF) patients. We have assessed whether this route of administration offers other benefits with regard to respiratory gene transfer. A standard jet nebuliser (Acorn System 22, Medicaid) was used to transfer the reporter gene beta-galactosidase complexed with the cationic liposome DC-Chol/DOPE to three epithelial cell lines in vitro, two non-CF and one CF, using a novel collection system. In all three cell lines, nebulisation resulted in significantly (P < 0.05) improved transfection efficiency compared with instillation. At a constant DNA: liposome ratio of 1:5 (wt:wt), transfection efficiency was inversely related to increasing concentrations of DNA-liposomes before nebulisation. This effect was not related to the amount of DNA delivered and measurements of both zeta potential and mean aerodynamic particle size before and after nebulisation did not show concentration-related differences. The increased transfection efficiency did not relate either to the physical consequences of the nebulisation processes nor the effects of nebulisation on the complexes before instillation. Significantly increased transfection efficiency was seen following nebulisation with 95% O2/5% CO2 in comparison with 21% O2/78% N2 (air); this did not relate to changes in either the pH or temperature of the solution bathing the cells. The data confirm that nebulisation is appropriate for gene delivery to the lower airways in clinical practice and points to factors that may optimise gene transfer efficiency.

Adenovirus vectors as transcomplementing templates for the production of replication defective retroviral vectors.

We have generated an adenovirus containing a retroviral vector sequence encoding the neomycin phosphotransferase (neo) gene (AV-LXSN). AV-LXSN transduction of retroviral packaging cell lines led to production of LXSN retroviral vector with alternative viral envelopes; exposure of target cells to retroviral containing supernatants confirmed envelope specific tropism. Retroviral titers (G418 cfu/ml) were comparable to those produced by standard techniques. Retrovirus could be detected in supernatants within 24 hours of AV-LXSN transduction and persisted as long as 120 hours. Southern blot analysis of DNA purified from populations of G418 cells showed the presence of a single neo containing restriction fragment of the appropriate size that could only be generated by reverse transcription of LXSN to produce LXSN provirus. This adeno-retroviral chimeric vector system could simplify the generation and testing of different retroviral vectors, particularly where assessment of vectors with alternative envelopes carrying novel targeting ligands is required.

The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro.

Trials of gene transfer for cystic fibrosis (CF) are currently underway. However, direct application to the airways may be impeded by the presence of airway secretions. We have therefore assessed the effect of CF sputum on the expression of the reporter gene beta-galactosidase complexed with the cationic liposome DC-Chol/DOPE in a number of cell lines in vitro. Transfection was markedly inhibited in the presence of sputum; the effect was concentration dependent and was only partially ameliorated by removal of sputum with phosphate-buffered saline (PBS) washing before gene transfer. However, treatment of the sputum-covered cells with recombinant human DNase (rhDNase, 50 micrograms/ml) but not with N-acetylcysteine, Nacystelyn, lysine (all 20 mM) or recombinant alginase (0.5 U/ml) significantly (P < 0.005) improved gene transfer. Adenovirus-mediated gene transfer efficiency in the presence of sputum was similarly inhibited, and again, treatment with rhDNase before transfection significantly improved gene transfer (P < 0.005). Transfection of Cos 7 cells in the presence of exogenous genomic DNA alone demonstrated similar inhibition to that observed with sputum and was also ameliorated by pre-treatment of DNA-covered cells with rhDNase. In a separate series of experiments performed in the absence of added sputum or genomic DNA, increasing concentrations of rhDNase resulted in a concentration-related decline in transfection efficiency. However, even at the highest concentration (500 micrograms/ml of rhDNase), transfection efficiency remained more than 50% of control. Thus, pre-treatment of CF airways with rhDNase may be appropriate before liposome or adenovirus-mediated gene therapy.

CFTR gene transfer reduces the binding of Pseudomonas aeruginosa to cystic fibrosis respiratory epithelium.

Much of the morbidity and mortality seen in cystic fibrosis (CF) is related to chronic infection of the respiratory tract with Pseudomonas aeruginosa. Some studies have attributed the strong relationship between CF and Pseudomonas colonization to the presence of increased numbers of specific cell-surface receptors, although other work suggests that this relates to the presence of mucus. Several groups are now assessing the use of gene transfer as a novel form of treatment for CF. We have examined whether P. aeruginosa binding to freshly obtained CF respiratory epithelial cells is increased, and have studied the effects of transfer of the CF transmembrane conductance regulator (CFTR) gene on this attachment. Binding of P. aeruginosa to noncultured nasal epithelial cells from both CF patients (n = 31) and healthy controls (n = 15) was studied with scanning electron microscopy. Binding was also assessed for CF cells following transfection with CFTR/liposome complexes. Epifluorescence microscopy was used to assess the effects of gene transfer on chloride fluxes. Adherence of P. aeruginosa directly to the cell surface of CF airway epithelium was significantly (P < 0.001) increased over that in non-CF controls. Liposome-mediated CFTR gene transfer resulted in a significant (P < 0.01) reduction in the numbers of bacteria bound to ciliated epithelial cells. Fluorescence microscopy confirmed correction of the basic chloride defect. Thus, in CF, the absence of normal CFTR results in increased binding of P. aeruginosa to respiratory epithelial cells. This abnormality can be corrected in vitro by restoration of CFTR function. This has important implications both for the pathogenesis of CF and for the future application and assessment of gene therapy for this disease.

Quantitative fluorescence measurements of chloride secretion in native airway epithelium from CF and non-CF subjects.

Functional assessment of the efficacy of CFTR gene transfer protocols in humans has previously involved measurement of in vivo potential difference. We have studied whether freshly obtained airway epithelial cells may provide suitable tissue for studies of in vivo gene transfer using fluorescent digital imaging microscopy. Nasal epithelial cells from non-cystic fibrosis subjects (n = 6) and from cystic fibrosis (CF) patients (delta F508: delta F508, n = 5) were obtained by brushing and loaded with 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Addition of the cAMP-agonists forskolin (20 microM) and 3-isobutyl-1-methylxanthine (IBMX, 100 microM) produced an increased efflux of iodide from the cells which was significantly (P < 0.05) greater in non-CF than in CF cells. Efflux following addition of the calcium ionophore, ionomycin (100 microM) was similar in both non-CF and CF cells. Liposome-mediated transfection of CF nasal epithelial cells in vitro with CFTR-cDNA restored the cAMP-stimulated efflux to non-CF values. Bronchial epithelial cells from non-CF subjects showed responses to forskolin and ionomycin that were not different to those in non-CF nasal epithelia. These data demonstrate that the assay provides a useful method for assessing correction of abnormal ion transport in non-cultured CF epithelium and is likely to provide a further assay for assessment of in vivo gene transfer efficiency in protocols of gene therapy for CF.

In vitro liposome-mediated DNA transfection of epithelial cell lines using the cationic liposome DC-Chol/DOPE.

Clinical trials using cationic liposome-mediated DNA transfer have now been initiated for several disorders including cystic fibrosis. Previous studies have shown that the level of gene expression achieved may be dependent on the formulation of the DNA-liposome complex and the cell type transfected. We have investigated, in vitro, the effect of parameters such as DNA:liposome ratio, dose and concentration on the level of transgene expression in epithelial cell lines using the cationic liposome DC-Chol/1,2-dioleoyl phosphatidylethanolamine (DOPE). A narrow range of conditions was found to produce maximal level of transgene expression within a particular cell line, as detected using the reporter molecule beta-galactosidase (beta-gal). beta-Gal expression was significantly enhanced by formulation of the DNA-DC-Chol/DOPE complexes in physiological solution at pH 9.0. Under standard in vitro transfection conditions, increased incubation time of the DNA-liposome complexes with cells resulted in increased transgene expression. In contrast, at relatively high DNA and liposome dose and concentrations, beta-gal activity was maximal after only 1 h of incubation, with a subsequent decrease in expression with time. The maximum level of expression that could be produced using fully optimised transfection conditions, however, was still highly dependent on each cell type analysed. Correlation of these findings with similar studies in vivo are now critical to determine the optimal formulation of DNA-liposome complexes for clinical application.

Bioelectric characteristics of exon 10 insertional cystic fibrosis mouse: comparison with humans.

Two important issues that can be addressed by animal models are disease pathogenesis and the testing of new treatments, including gene therapy. How closely these models mimic the relevant disorder in humans will determine their usefulness. This study examines how closely the characteristic bioelectric features of cystic fibrosis (CF) are reproduced in the airways and intestinal tract of the exon 10 insertional mutant mouse (cf/cf). In agreement with CF subjects these cf/cf mutant mice demonstrate the following: 1) reduced adenosine 3',5'-cyclic monophosphate-related chloride secretion throughout the respiratory and intestinal tracts both in vivo and in vitro, 2) calcium-related chloride secretion that is preserved in the airways but reduced in the intestine, and 3) a more negative nasal potential difference and increased amiloride response compared with wild-type animals, likely to relate to increased sodium absorption. In contrast to humans, sodium absorption is not increased in the small intestine and is reduced in the trachea of the cf/cf mice. We conclude that the majority of the salient electrophysiological features of CF required for studies of pathogenesis or testing of new treatments are present in these cf/cf mice.

Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis.

We report the results of a double-blind, placebo-controlled trial in nine cystic fibrosis (CF) subjects receiving cationic liposome complexed with a complementary DNA encoding the CF transmembrane conductance regulator (CFTR), and six CF subjects receiving only liposome to the nasal epithelium. No adverse clinical effects were seen and nasal biopsies showed no histological or immuno-histological changes. A partial restoration of the deficit between CF and non-CF subjects of 20% was seen for the response to low Cl- perfusion following CFTR cDNA administration. This was maximal around day three and had reverted to pretreatment values by day seven. In some cases the response to low Cl- was within the range for non-CF subjects. Plasmid DNA and transgene-derived RNA were detected in the majority of treated subjects. Although these data are encouraging, it is likely that transfection efficiency and the duration of expression will need to be increased for therapeutic benefit.

Gene therapy for cystic fibrosis in humans by liposome-mediated DNA transfer: the production of resources and the regulatory process.

The number of clinical trials using gene transfer technology, either active or under discussion, is increasing rapidly. However, little information is available describing the regulatory procedures or safety specifications that must be considered before initiation of such trials in Europe. We describe the procedure used by our group to produce resources for the first stage of a phase I trial of liposome-mediated gene therapy for cystic fibrosis. The current lack of written and co-ordinated guidance from the numerous interested regulatory agencies within the UK and Europe makes determination of the appropriate safety specifications and procedures for these novel trials difficult, as does the fact that some new agencies (such as the Genetic Therapy Advisory Committee in the UK) and some which are unfamiliar with clinical trials (such as the Department of the Environment) are involved as well as the Medicines Control Agency. In addition, we estimate that the realistic cost of these trials, which in many cases will have to be covered from research budgets provided by government agencies or medical charities, could lead to delays in the clinical application of this important new therapeutic strategy.

Nasal application of the cationic liposome DC-Chol:DOPE does not alter ion transport, lung function or bacterial growth.

Liposome-mediated gene transfer is commonly used for in vitro transfection of deoxyribonucleic acid (DNA) into mammalian cells. We and others have recently demonstrated that this can be an effective method for in vivo delivery of plasmid DNA containing the human cystic fibrosis transmembrane conductance regulator (CFTR) gene to mouse models of cystic fibrosis (CF). This suggests that cationic liposomes may be useful for transferring CFTR complementary DNA (cDNA) into the airways of CF subjects. In such trials, measurement of nasal potential difference (PD) will be used to monitor the efficacy of correction of the CF bioelectric defect and to provide a sensitive assay of epithelial integrity [corrected]. We therefore assessed whether the cationic liposome DC-Chol: DOPE altered nasal ion transport parameters, in six normal and three CF subjects. Lung function was also measured as a further marker of safety. Finally, as CF airways are chronically infected, we studied whether DC-Chol:DOPE or DC-Chol:DOPE-DNA complexes altered the bacterial growth and sensitivities of CF sputum. No significant effect was seen on any of these parameters, suggesting that DC-Chol:DOPE may be appropriate for use in human trials of liposome-mediated gene therapy for CF.