Natasha J. Caplen

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

Short interfering RNAs (siRNAs) are double-stranded RNAs of ≈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.

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.

Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells

RNA interference (RNAi) mediated by short interfering RNAs (siRNAs) is a widely used method to analyze gene function. To use RNAi knockdown accurately to infer gene function, it is essential to determine the specificity of siRNA-mediated RNAi. We have assessed the specificity of 10 different siRNAs corresponding to the MEN1 gene by examining the expression of two additional genes, TP53 (p53) and CDKN1A (p21), which are considered functionally unrelated to menin but are sensitive markers of cell state. MEN1 RNA and corresponding protein levels were all reduced after siRNA transfection of HeLa cells, although the degree of inhibition mediated by individual siRNAs varied. Unexpectedly, we observed dramatic and significant changes in protein levels of p53 and p21 that were unrelated to silencing of the target gene. The modulations in p53 and p21 levels were not abolished on titration of the siRNAs, and similar results were obtained in three other cell lines; in none of the cell lines tested did we see an effect on the protein levels of actin. These data suggest that siRNAs can induce nonspecific effects on protein levels that are siRNA sequence dependent but that these effects may be difficult to detect until genes central to a pivotal cellular response, such as p53 and p21, are studied. We find no evidence that activation of the double-stranded RNA-triggered IFN-associated antiviral pathways accounts for these effects, but we speculate that partial complementary sequence matches to off-target genes may result in a micro-RNA-like inhibition of translation.

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.

RNAi as a gene therapy approach

RNA duplexes of 21 – 23 nucleotides (nts), with ~ 2 nt 3’ overhangs (called small interfering RNAs or siRNAs), have recently been shown to mediate sequence-specific inhibition of gene expression in mammalian cells via a post-transcriptional gene silencing (PTGS) mechanism termed RNA interference (RNAi). RNAi has been rapidly adopted as a functional genomics tool in a wide range of species, has been adapted to allow for the transient or stable knockdown of gene expression generation in cell lines and animals, and has been developed for high-throughput analysis of gene function in Caenorhabditis elegans. With an increasing list of genes successfully knocked-down by RNAi in mammalian cells and improvements in the delivery of siRNAs to cells, including in vivo delivery to mice, attention is now turning to assessing the potential RNAi has as a gene therapy approach. RNAi is likely to have the greatest impact as a therapeutic tool in two key clinical areas, cancer and infectious disease, but it also has the potential as a therapy for other disorders including some dominant genetic diseases. This review will describe the status of the science behind this novel mechanism and will illustrate the therapeutic potential of RNAi-based technologies, using examples from these critical clinical research areas.

Selective Toxicity of NSC73306 in MDR1-Positive Cells as a New Strategy to Circumvent Multidrug Resistance in Cancer

ATP-binding cassette (ABC) proteins include the best known mediators of resistance to anticancer drugs. In particular, ABCB1 [MDR1/P-glycoprotein (P-gp)] extrudes many types of drugs from cancer cells, thereby conferring resistance to those agents. Attempts to overcome P-gp-mediated drug resistance using specific inhibitors of P-gp has had limited success and has faced many therapeutic challenges. As an alternative approach to using P-gp inhibitors, we characterize a thiosemicarbazone derivative (NSC73306) identified in a generic screen as a compound that exploits, rather than suppresses, P-gp function to induce cytotoxicity. Cytotoxic activity of NSC73306 was evaluated in vitro using human epidermoid, ovarian, and colon cancer cell lines expressing various levels of P-gp. Our findings suggest that cells become hypersensitive to NSC73306 in proportion to the increased P-gp function and multidrug resistance (MDR). Abrogation of both sensitivity to NSC73306 and resistance to P-gp substrate anticancer agents occurred with specific inhibition of P-gp function using either a P-gp inhibitor (PSC833, XR9576) or RNA interference, suggesting that cytotoxicity was linked to MDR1 function, not to other, nonspecific factors arising during the generation of resistant or transfected cells. Molecular characterization of cells selected for resistance to NSC73306 revealed loss of P-gp expression and consequent loss of the MDR phenotype. Although hypersensitivity to NSC73306 required functional expression of P-gp, biochemical assays revealed no direct interaction between NSC73306 and P-gp. This article shows that NSC73306 kills cells with intrinsic or acquired P-gp-induced MDR and indirectly acts to eliminate resistance to MDR1 substrates.

Kinase-Independent Functions for Itk in TCR-Induced Regulation of Vav and the Actin Cytoskeleton

The Tec family kinase Itk is an important regulator of Ca2+ mobilization and is required for in vivo responses to Th2-inducing agents. Recent data also implicate Itk in TCR-induced regulation of the actin cytoskeleton. We have evaluated the requirements for Itk function in TCR-induced actin polarization. Reduction of Itk expression via small interfering RNA treatment of the Jurkat human T lymphoma cell line or human peripheral blood T cells disrupted TCR-induced actin polarization, a defect that correlated with decreased recruitment of the Vav guanine nucleotide exchange factor to the site of Ag contact. Vav localization and actin polarization could be rescued by re-expression of either wild-type or kinase-inactive murine Itk but not by Itk containing mutations affecting the pleckstrin homology or Src homology 2 domains. Additionally, we find that Itk is constitutively associated with Vav. Loss of Itk expression did not alter gross patterns of Vav tyrosine phosphorylation but appeared to disrupt the interactions of Vav with SLP-76. Expression of membrane-targeted Vav, Vav-CAAX, can rescue the small interfering RNA to Itk-induced phenotype, implicating the alteration in Vav localization as directly contributing to the actin polarization defect. These data suggest a kinase-independent scaffolding function for Itk in the regulation of Vav localization and TCR-induced actin polarization.

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 β-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 μg/ml) but not with N-acetylcysteine, Nacystelyn, lysine (all 20 m&mcy;) 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 μg/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.

The identification of microRNAs in a genomically unstable region of human chromosome 8q24.

The PVT1 locus is identified as a cluster of T(2;8) and T(8;22) “variant” MYC-activating chromosomal translocation breakpoints extending 400 kb downstream of MYC in a subset (≈20%) of Burkitts lymphoma (vBL). Recent reports that microRNAs (miRNA) may be associated with fragile sites and cancer-associated genomic regions prompted us to investigate whether the PVT1 region on chromosome 8q24 may contain miRNAs. Computational analysis of the genomic sequence covering the PVT1 locus and experimental verification identified seven miRNAs. One miRNA, hsa-miR-1204, resides within a previously described PVT1 exon (1b) that is often fused to the immunoglobulin light chain constant region in vBLs and is present in high copy number in MYC/PVT1–amplified tumors. Like its human counterpart, mouse mmu-miR-1204 represents the closest miRNA to Myc (∼50 kb) and is found only 1 to 2 kb downstream of a cluster of retroviral integration sites. Another miRNA, mmu-miR-1206, is close to a cluster of variant translocation breakpoints associated with mouse plasmacytoma and exon 1 of mouse Pvt1. Virtually all the miRNA precursor transcripts are expressed at higher levels in late-stage B cells (including plasmacytoma and vBL cell lines) compared with immature B cells, suggesting possible roles in lymphoid development and/or lymphoma. In addition, lentiviral vector-mediated overexpression of the miR-1204 precursor (human and mouse) in a mouse pre–B-cell line increased expression of Myc. High levels of expression of the hsa-miR-1204 precursor is also seen in several epithelial cancer cell lines with MYC/PVT1 coamplification, suggesting a potentially broad role for these miRNAs in tumorigenesis. (Mol Cancer Res 2008;6(2):212–21)

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.