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PubMed2006

Can siRNA technology provide the tools for gene therapy of the future?

A new era in genetics has started 15 years ago, when co-suppression in petunia has been discovered. Later, co-suppression was identified as RNA interference (RNAi) in many plant and lower eukaryote animals. Although an ancient antiviral host defense mechanism in plants, the physiologic role of RNAi in mammals is still not completely understood. RNAi is directed by short interfering RNAs (siRNAs), one subtype of short double stranded RNAs. In this review we summarize the history and mechanisms of RNAi. We also aim to highlight the correlation between structure and efficacy of siRNAs. Delivery is the most important obstacle for siRNA based gene therapy. Viral and nonviral deliveries are discussed. In vivo delivery is the next obstacle to clinical trials with siRNAs. Although hydrodynamic treatment is effective in animals, it cannot be used in human therapy. One possibility is organ selective catheterization. The known side effects of synthesized siRNAs are also discussed. Although there are many problems to face in this new field of gene therapy, successful in vitro and in vivo experiments raise hope for treating human disease with siRNA.

Genetic Therapy↗
PubMed2004

A plasmid-based system for expressing small interfering RNA libraries in mammalian cells.

BACKGROUND: RNA interference (RNAi) is an evolutionarily conserved process that functions to inhibit gene expression. The use of RNAi in mammals as a tool to study gene function has rapidly developed in the last couple of years since the discovery that the function-inhibiting units of RNAi are short 21-25 nt double-stranded RNAs (siRNAs) derived from their longer template. The use of siRNAs allows for gene-specific knock-down without induction of the non-specific interferon response in mammalian cells. Multiple systems have been developed to introduce siRNAs into mammals. One of the most appealing of these techniques is the use of vectors containing polymerase III promoters to drive expression of hairpin siRNAs. However, there are multiple limitations to using hairpin siRNA vectors including the observation that some are unstable in bacteria and are difficult to sequence. RESULTS: To circumvent the limitation of hairpin siRNA vectors we have developed a convergent opposing siRNA expression system called pHippy. We have generated pHippy vectors or expression cassettes that knock down the expression of both reporter and endogenous genes. As a proof of principle that pHippy can be used to generate random siRNA libraries, we generated a small siRNA library against PGL3 luciferase and demonstrated that we could recover functional siRNAs that knock down PGL3 luciferase. CONCLUSIONS: siRNA is a powerful tool to study gene function. We have developed a new vector with opposing convergent promoters for the expression of siRNAs, which can be used to knock down endogenous genes in a high throughput manner or to perform functional screening with random or cDNA-derived siRNA libraries.

Animals↗
PubMed2005

Downregulation of the nuclear-encoded subunits of the complexes III and IV disrupts their respective complexes but not complex I in procyclic Trypanosoma brucei.

The function, stability and mutual interactions of selected nuclear-encoded subunits of respiratory complexes III and IV were studied in the Trypanosoma brucei procyclics using RNA interference (RNAi). The growth rates and oxygen consumption of clonal cell lines of knock-downs for apocytochrome c1 (apoc1) and the Rieske Fe-S protein (Rieske) of complex III, and cytochrome c oxidase subunit 6 (cox6) of complex IV were markedly decreased after RNAi induction. Western analysis of mitochondrial lysates using specific antibodies confirmed complete elimination of the targeted proteins 4-6 days after induction. The Rieske protein was reduced in the apoc1 knock-down and vice versa, indicating a mutual interdependence of these components of complex III. However, another subunit of complex IV remained at the wild-type level in the cox6 knock-down. As revealed by two-dimensional blue native/SDS-PAGE electrophoresis, silencing of a single subunit resulted in the disruption of the respective complex, while the other complex remained unaffected. Membrane potential was reproducibly decreased in the knock-downs and the activities of complex III and/or IV, but not complex I, were drastically reduced, as measured by activity assays and histochemical staining. Using specific inhibitors, we have shown that in procyclics with depleted subunits of the respiratory complexes the flow of electrons was partially re-directed to the alternative oxidase. The apparent absence in T. brucei procyclics of a supercomplex composed of complexes I and III may represent an ancestral state of the respiratory chain.

Animals↗
PubMed2003

Integrins and EGFR coordinately regulate the pro-apoptotic protein Bim to prevent anoikis.

Epithelial cells must adhere to the extracellular matrix (ECM) for survival, as detachment from matrix triggers apoptosis or anoikis. Integrins are major mediators of adhesion between cells and ECM proteins, and transduce signals required for cell survival. Recent evidence suggests that integrin receptors are coupled to growth factor receptors in the regulation of multiple biological functions; however, mechanisms involved in coordinate regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we identify the pro-apoptotic protein Bim as a critical mediator of anoikis in epithelial cells. Bim is strongly induced after cell detachment and downregulation of Bim expression by RNA interference (RNAi) inhibits anoikis. Detachment-induced expression of Bim requires a lack of beta(1)-integrin engagement, downregulation of EGF receptor (EGFR) expression and inhibition of Erk signalling. Overexpressed EGFR was uncoupled from integrin regulation, resulting in the maintenance of Erk activation in suspension, and a block in Bim expression and anoikis. Thus, Bim functions as a key sensor of integrin and growth factor signals to the Erk pathway, and loss of such coordinate regulation may contribute to tumour progression.

Animals↗
PubMed2006

Abundant transcripts from retrotransposons are unstable in fully grown mouse oocytes.

One physiological function proposed for RNA interference (RNAi) is to constrain expression of repetitive elements and thereby reduce the incidence of retrotransposition. Consistent with this model is that inhibiting the RNAi pathway results in an increase in expression of repetitive elements in preimplantation mouse embryos. Mouse oocytes are essentially transcriptionally quiescent providing a unique opportunity to assess the stability of repetitive element-derived transcripts in these cells. We compared the transcriptome of freshly isolated fully grown germinal vesicle (GV)-intact oocytes to that of oocytes in which meiotic maturation in vitro was inhibited for 48 h by milrinone. Consistent with the aforementioned function for RNAi is that the abundance of only a relatively small number of transcripts decreased in the cultured oocytes, when compared to changes that occur during maturation or following fertilization, and of those, several belonged to mobile elements.

Animals↗
PubMed2005

Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors.

BACKGROUND: There has been much research into the use of RNA interference (RNAi) for the treatment of human diseases. Many viruses, including hepatitis B virus (HBV), are susceptible to inhibition by this mechanism. However, for RNAi to be effective therapeutically, a suitable delivery system is required. METHODS: Here we identify an RNAi sequence active against the HBV surface antigen (HBsAg), and demonstrate its expression from a polymerase III expression cassette. The expression cassette was inserted into two different vector systems, based on either prototype foamy virus (PFV) or adeno-associated virus (AAV), both of which are non-pathogenic and capable of integration into cellular DNA. The vectors containing the HBV-targeted RNAi molecule were introduced into 293T.HBs cells, a cell line stably expressing HBsAg. The vectors were also assessed in HepG2.2.15 cells, which secrete infectious HBV virions. RESULTS: Seven days post-transduction, a knockdown of HBsAg by approximately 90%, compared with controls, was detected in 293T.HBs cells transduced by shRNA encoding PFV and AAV vectors. This reduction has been observed up to 5 months post-transduction in single cell clones. Both vectors successfully inhibited HBsAg expression from HepG2.2.15 cells even in the presence of HBV replication. RT-PCR of RNA extracted from these cells showed a reduction in the level of HBV pre-genomic RNA, an essential replication intermediate and messenger RNA for HBV core and polymerase proteins, as well as the HBsAg messenger RNA. CONCLUSIONS: This work is the first to demonstrate that delivery of RNAi by viral vectors has therapeutic potential for chronic HBV infection and establishes the ground work for the use of such vectors in vivo.

Base Sequence↗
PubMed2004

ROLL: a method of preparation of gene-specific oligonucleotide libraries.

The selection of nucleic acid sequences capable of specifically and efficiently hybridizing to target sequences is crucial to the success of many applications, including microarrays, PCR and other amplification procedures, antisense inhibition, ribozyme-mediated cleavage, and RNA interference (RNAi). Methods of selection using nucleotide sequence libraries have several advantages over rational approaches using defined sequences. However, the high complexity of completely random (degenerate) libraries and their high toxicity in cell-based assays make their use in many applications impractical. Gene-specific oligonucleotide libraries, which contain all possible sequences of a certain length occurring within a given gene, have much lower complexity and, thus, can significantly simplify and accelerate sequence screening. Here, we describe a new method for the preparation of gene-specific libraries using the ligation of randomized oligonucleotide probes hybridized adjacently on target polynucleotide templates followed by PCR amplification. We call this method random oligonucleotide ligated libraries (ROLL).

Base Sequence↗
PubMed2005

Immune responses to dsRNA: implications for gene silencing technologies.

Nucleic acid-induced gene silencing, such as RNA interference (RNAi), induces a multitude of responses in addition to the knockdown of a gene. This is best understood in the context of the antiviral immune response, from which the processes of RNAi are thought to be derived. Viral challenge of a vertebrate host leads to an intricate series of responses that orchestrate antiviral immunity. The success of this multifaceted system in overcoming viral encounters hinges on complex pathogen-host interactions. One aspect of these interactions, the nucleic acid-based immune response, is key to the successful resolution of a viral challenge. In particular, dsRNA, a nucleic acid associated with viral replication, is involved in numerous interactions contributing to induction, activation and regulation of antiviral mechanisms. Specifically, dsRNA is responsible for stimulating important protective responses, such as the activation of dicer-related antiviral pathways, induction of type 1 IFN, and stimulation of dsRNA-activated protein kinase and oligoadenylate synthetase. Furthermore, the modulation and shaping of this overall immune response is facilitated through nucleic acid interactions with pattern recognition receptors such as toll-like receptor 3. These diverse dsRNA-induced antiviral responses have implications for biotechnologies that use dsRNA to harness one arm of the host antiviral machinery for silencing a specific target gene. The interlinked nature of these response elements means that it may be difficult to completely isolate one element from the other arms of the antiviral response program of an organism. Thus, it is beneficial to understand all aspects of the immune response to dsRNA in order to manipulate these systems and minimize unwanted non-specific effects.

Animals↗
PubMed2004

siRNA cell arrays for high-content screening microscopy.

RNA interference (RNAi) is a recent advance that provides the possibility to reduce the expression of specific target genes in cultured mammalian cells with potential applications on a genome-wide scale. However, to achieve this, robust methodologies that allow automated and efficient delivery of small interfering RNAs (siRNAs) into living cultured cells and reliable quality control of siRNA function must be in place. Here we describe the production of cell arrays for reverse transfection of tissue culture cells with siRNA and plasmid DNA suitable for subsequent high-content screening microscopy applications. All the necessary transfection components are mixed prior to the robotic spotting on noncoated chambered coverglass tissue culture dishes, which are ideally suited for time-lapse microscopy applications in living cells. The addition of fibronectin to the spotting solution improves cell adherence. After cell seeding, no further cell culture manipulations, such as medium changes or the addition of 7 serum, are needed. Adaptation of the cell density improves autofocus performance for high-quality data acquisition and cell recognition. The co-transfection of a nonspecific fluorescently labeled DNA oligomer with the specific siRNA helps to mark each successfully transfected cell and cell cluster. We demonstrate such an siRNA cell array in a microscope-based functional assay in living cells to determine the effect of various siRNA oligonucleotides against endogenous targets on cellular secretion.

Cell Line↗
PubMed2003

siRNA targeting of the viral E6 oncogene efficiently kills human papillomavirus-positive cancer cells.

The targeted inhibition of antiapoptotic factors in tumour cells may provide a rational approach towards the development of novel anticancer therapies. Using human papillomavirus (HPV)-transformed cells as a model system, we investigated if RNA interference (RNAi)-mediated gene silencing can be employed in order to overcome the apoptosis resistance of cancer cells. We found that both vector-borne and synthetic small interfering (si)RNAs, specifically directed against the antiapoptotic HPV E6 oncogene, restored dormant tumour suppressor pathways in HPV-positive cancer cells that are otherwise inactive in the presence of E6. This ultimately resulted in massive apoptotic cell death, selectively in HPV-positive tumour cells. These findings show that RNAi provides a powerful molecular strategy to inactivate intracellular E6 function efficiently. Moreover, they define E6 as a most promising therapeutic target to eliminate HPV-positive tumour cells specifically by RNAi. Thus, by sequence-specific targeting of antiapoptotic genes, siRNAs may be developed into novel therapeutics that can efficiently correct the apoptosis deficiency of cancer cells.

Apoptosis↗
PubMed2005

Inhibition of Measles virus multiplication in cell culture by RNA interference.

Measles takes a toll of one million people a year worldwide, especially in developing countries. As the so far applied vaccines were not able to control this disease, novel approaches to this problem are needed. In this study, we investigated the possibility of inhibition of Measles virus (MV) infection through RNA interference (RNAi), namely the interference with the second but main receptor of MV, signaling lymphocyte activation molecule (SLAM), in an Epstein-Barr virus (EBV)-transformed marmoset B cell line B95-8. Three potential target sequences in the SLAM gene were chosen and cloned into the pSilencer 3.0-H1 vector. The vector constructs were used for transfection of B95-8 cells in which SLAM was then detected by flow cytometry, Western blot analysis and RT-PCR both at transcription and expression levels. One of the vector constructs was found to block effectively the SLAM expression as well as MV infection. These results suggest that RNAi could silence SLAM expression and inhibit the MV infection of host cells.

Amino Acid Sequence↗
PubMed2006

Glycogen synthase kinase-3 is a negative regulator of extracellular signal-regulated kinase.

Glycogen-synthase kinase-3 (GSK-3) and extracellular signal-regulated kinase (ERK) are critical downstream signaling proteins for the PI3-kinase/Akt and Ras/Raf/MEK-1 pathway, respectively, and regulate diverse cellular processes including embryonic development, cell differentiation and apoptosis. Here, we show that inhibition of GSK-3 using GSK-3 inhibitors or RNA interference (RNAi) significantly induced the phosphorylation of ERK1/2 in human colon cancer cell lines HT29 and Caco-2. Pretreatment with the PKCdelta-selective inhibitor rottlerin or transfection with PKCdelta siRNA attenuated the phosphorylation of ERK1/2 induced by the GSK-3 inhibitor SB-216763 and, furthermore, treatment with SB-216763 or transfection with GSK-3alpha and GSK-3beta siRNA increased PKCdelta activity, thus identifying a role for PKCdelta in the induction of ERK1/2 phosphorylation by GSK-3 inhibition. Treatment with SB-216763 increased expression of cyclooxygenase-2 (COX-2) and IL-8, which are downstream targets of ERK1/2 activation; this induction was abolished by MEK/ERK inhibition, suggesting GSK-3 inhibition induced COX-2 and IL-8 through ERK1/2 activation. The transcriptional induction of COX-2 and IL-8 by GSK-3 inhibition was further demonstrated by the increased COX-2 and IL-8 promoter activity after SB-216763 treatment or transfection with GSK-3alpha or GSK-3beta siRNA. Importantly, our findings identify GSK-3, acting through PKCdelta, as a negative regulator of ERK1/2, thus revealing a novel crosstalk mechanism between these critical signaling pathways.

Apoptosis↗
PubMed2004

Defective mitochondrial protein translocation precludes normal Caenorhabditis elegans development.

We demonstrate biochemically that the genes identified by sequence similarity as orthologs of the mitochondrial import machinery are functionally conserved in Caenorhabditis elegans. Specifically, tin-9.1 and tin-10 RNA interference (RNAi) treatment of nematodes impairs import of the ADP/ATP carrier into isolated mitochondria. Developmental phenotypes are associated with gene knock-down of the mitochondrial import components. RNAi of tomm-7 and ddp-1 resulted in mitochondria with an interconnected morphology in vivo, presumably due to defects in the assembly of outer membrane fission/fusion components. RNAi of the small Tim proteins TIN-9.1, TIN-9.2, and TIN-10 resulted in a small body size, reduced number of progeny produced, and partial embryonic lethality. An additional phenotype of the tin-9.2(RNAi) animals is defective formation of the somatic gonad. The biochemical demonstration that the protein import activity is reduced, under the same conditions that yield the defects in specific tissues and lethality in a later generation, suggests that the developmental abnormalities observed are a consequence of defects in mitochondrial inner membrane biogenesis.

Animals↗
PubMed2006

The cyclin-dependent kinase (CDK) family member PNQALRE/CCRK supports cell proliferation but has no intrinsic CDK-activating kinase (CAK) activity.

The cyclin-dependent kinases (CDKs) that drive the eukaryotic cell cycle must be phosphorylated within the activation segment (T-loop) by a CDK-activating kinase (CAK) to achieve full activity. Although a requirement for CDK-activating phosphorylation is conserved throughout eukaryotic evolution, CAK itself has diverged between metazoans and budding yeast, and fission yeast has two CAKs, raising the possibility that additional mammalian enzymes remain to be identified. We report here the characterization of PNQALRE (also known as CCRK or p42), a member of the mammalian CDK family most similar to the cell-cycle effectors Cdk1 and Cdk2 and to the CAK, Cdk7. Although PNQALRE/CCRK was recently proposed to activate Cdk2, we show that the monomeric protein has no intrinsic CAK activity. Depletion of PNQALRE by >80% due to RNA interference (RNAi) impairs cell proliferation, but fails to arrest the cell cycle at a discrete point. Instead, both the fraction of cells with a sub-G(1) DNA content and cleavage of poly(ADP-ribose) polymerase (PARP) increase. PNQALRE knockdown did not diminish Cdk2 T-loop phosphorylation in vivo or decrease CAK activity of a cell extract. In contrast, depletion of Cdk7 by RNAi causes a proportional decrease in the ability of an extract to activate recombinant Cdk2. Our data do not support the proposed function of PNQALRE/CCRK in activating CDKs, but instead reinforce the notion of Cdk7 as the major, and to date the only, CAK in mammalian cells.

Amino Acid Sequence↗
PubMed2006

Cortactin affects cell migration by regulating intercellular adhesion and cell spreading.

Cortactin is an F-actin binding protein that stabilizes F-actin networks and promotes actin polymerization by activating the Arp2/3 complex. Overexpression of cortactin, as observed in several human cancers, stimulates cell migration, invasion, and experimental metastasis; however, the underlying mechanism is not understood. To investigate the importance of cortactin in cell migration, we downregulated its expression using RNA interference (RNAi). Stable downregulation of cortactin in HBL100 breast epithelial cells resulted in (i) decreased cell migration and invasion, (ii) enhanced cell-cell adhesion, and (iii) accelerated cell spreading. These phenotypic changes were reversed by expression of RNAi-resistant mouse cortactin. Cortactin colocalized with cadherin and beta-catenin in adherens junctions, consistent with its role in intercellular adhesion. Remarkably, cortactin deficiency did not affect lamellipodia formation. Instead, downregulation of cortactin in human squamous carcinoma cells that overexpress cortactin changed the cytoskeletal organization. We conclude that increased levels of cortactin, as found in human carcinomas, promote cell migration and invasion by reducing cell spreading and intercellular adhesive strength.

Actin-Related Protein 2-3 Complex↗
PubMed2003

RNA interference in the moss Physcomitrella patens.

The moss Physcomitrella patens performs efficient homologous recombination, which allows for the study of individual gene function by generating gene disruptions. Yet, if the gene of study is essential, gene disruptions cannot be isolated in the predominantly haploid P. patens. Additionally, disruption of a gene does not always generate observable phenotypes due to redundant functions from related genes. However, RNA interference (RNAi) can provide mutants for both of these situations. We show that RNAi disrupts gene expression in P. patens, adding a significant tool for the study of plant gene function. To assay for RNAi in moss, we constructed a line (NLS-4) expressing a nuclearly localized green fluorescent protein (GFP):beta-glucuronidase (GUS) fusion reporter protein. We targeted the reporter protein with two RNAi constructs, GUS-RNAi and GFP-RNAi, expressed transiently by particle bombardment. Transformed protonemal cells are marked by cobombardment with dsRed2, which diffuses between the nucleus and cytoplasm. Cells transformed with control constructs have nuclear/cytoplasmic red fluorescence and nuclear green fluorescence. In cells transformed with GUS-RNAi or GFP-RNAi constructs, the nuclear green fluorescence was reduced on average 9-fold as soon as 48 h after transformation. Moreover, isolated lines of NLS-4 stably transformed with GUS-RNAi construct have silenced nuclear GFP, indicating that RNAi is propagated stably. Thus, RNAi adds a powerful tool for functional analysis of plant genes in moss.

Bryopsida↗
PubMed2001

Multiple interactions between SRm160 and SR family proteins in enhancer-dependent splicing and development of C. elegans.

BACKGROUND: SR family and SR-related proteins assemble on exonic splicing enhancer (ESE) sequences to promote both constitutive and regulated splicing. The SRm160 splicing coactivator, an SR-related nuclear matrix protein of 160 kDa, is important for the splicing of specific constitutive and ESE-dependent pre-mRNAs. RESULTS: In the present study, we show that SRm160 is required to promote pre-mRNA splicing mediated by a large population of functional ESE sequences within a randomized 18 nucleotide sequence. This suggests that it functions as a general coactivator by interacting with different SR family/SR-related proteins bound to different ESE sequences. Consistent with this, several SR family and SR-related proteins coimmunoprecipitated specifically with SRm160 in the presence of low salt. We used RNA interference (RNAi) in Caenorhabditis elegans to determine whether interactions between CeSRm160 and different CeSR family proteins are important in a whole-organism context. Previously we showed that RNAi of CeSRm160 and individual CeSR family genes other than CeSF2/ASF results in no obvious phenotype, which is indicative of gene redundancy. In the present study, we demonstrate that RNAi of CeSRm160 in combination with any CeSR family gene results in the production of unfertilized oocytes by the injected mother. CONCLUSIONS: The observation that simultaneous suppression of CeSRm160 and individual CeSR family proteins results in a distinct phenotype is indicative of critical functional interactions between these factors. Our results provide biochemical and genetic evidence indicating that interactions between SRm160 and multiple SR family proteins are important for both optimal splicing activity and for proper development.

Amino Acid Sequence↗
PubMed2003

RNA interference-mediated silencing of the fatty acid synthase gene attenuates growth and induces morphological changes and apoptosis of LNCaP prostate cancer cells.

Fatty acid synthase (FASE), a key enzyme in the biosynthesis of fatty acids, is markedly overexpressed in many human epithelial cancers, rendering it an interesting target for antineoplastic therapy. Here, using the potent and highly sequence-specific mechanism of RNA interference (RNAi), we have silenced the expression of FASE in lymph node carcinoma of the prostate (LNCaP) cells. RNAi-mediated down-regulation of FASE expression resulted in a major decrease in the synthesis of triglycerides and phospholipids and induced marked morphological changes, including a reduction in cell volume, a loss of cell-cell contacts, and the formation of spider-like extrusions. Furthermore, silencing of the FASE gene by RNAi significantly inhibited LNCaP cell growth and ultimately resulted in induction of apoptosis. Importantly and in striking contrast with LNCaP cells, RNAi-mediated inhibition of FASE did not influence growth rate or viability of nonmalignant cultured skin fibroblasts. These data indicate that RNAi opens new avenues toward the study of the role of FASE overexpression in tumor cells and provides an interesting and selective alternative to chemical FASE inhibitors in the development of antineoplastic therapy.

Apoptosis↗