Application Of Gene Silencing Research Articles

Abstract 1172: The impact of siRNA targeting Colon Cancer Associated Transcript 1 (CCAT-1) on proliferation of HT-29 cells

Abstract Introduction: Colon cancer Associated Transcript-1 (CCAT-1) is a non-coding RNA transcript highly expressed in colorectal cancer (CRC) but not in normal colonic tissue. The role of CCAT-1 in tumorigenesis is yet to be defined. Small interfering RNAs (siRNA) are a group of sequence-specific posttranscriptional gene silencing molecules. Recent advances in the design and delivery of targeting siRNA molecules allows efficient and highly specific gene silencing in mammalian systems. The most common application of siRNA gene-silencing in oncology is to identify loss-of-function phenotypes in genes that will result in decreased proliferation or death of cancer cells. Aim: To study the role of colon cancer associated transcript 1 (CCAT-1) in proliferation of human colon cancer cells using RNA interference. Methods: Small interfering RNAs (siRNA) targeting CCAT-1 were transfected into HT-29 colon cancer cells. At day one, 80×105 HT-29 cells per well were plated and suspended in RPMi 1640 medium. At day two, 50 pmol CCAT-1 siRNA and 0.5 µl lipofectamine 2000 were added to each well. CCAT-1 expression was studied by real-time PCR in order to evaluate gene silencing. Cell proliferation was studied by MTT incorporation assay. Twenty four hrs after transfection, 50 µl of MTT were added to the cells media and incubated for 4 h at 37° C. The purple Formazan product, converted MTT, was dissolved by the addition of 150µl/well DMSO and OD readings were obtained at 550 nm. Results:By examining AV average OD readings at 550 nm, untreated HT-29 cells (10 wells), cells treated with lipofectamine 2000 (10 wells) and cells silenced by siRNA of CCAT-1 (10 wells) showed a significant decrease in proliferation: 2.4±0.64, 1.54±0.63, 0.8±0.14 respectively (p=0.002). Furthermore, comparing AV OD readings between 24 and 48 h in the silenced cells, showed a decrease of 38% in proliferation. This was compared to untransfected cells and cells treated with lipofectamine 2000 which showed an increase of 39% and 22% respectively. Twenty four hrs after transfection, real time PCR results showed a reduction in CCAT-1 RNA levels of 70% compared to controls and 48 hrs after transfection, CCAT-1 RNA levels were reduced to 55% of baseline. Conclusions: Our results may support a role for CCAT-1 in the regulation of colon cancer cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1172. doi:10.1158/1538-7445.AM2011-1172

Polyamine functionalized carbon nanotubes: synthesis, characterization, cytotoxicity and siRNA binding

In this work we have synthesized a new series of cationic carbon nanotubes (CNTs) for siRNA binding. Both single- and multi-walled CNTs have been modified by addition or amidation reaction with short linear polyamine chains including putrescine, spermidine and spermine. All the new conjugates have been characterized with several spectroscopic and microscopic techniques. Their cytotoxic effects have been assessed on human lung carcinoma cell line. Finally, we have analyzed their capacity to bind siRNA towards the development of new carriers for gene silencing applications. The dispersibility properties and the capacity to complex siRNA of the different conjugates were found to be strongly dependent on the position of the functional groups on CNTs (i.e. mainly at the tips following the amidation reaction or on the sidewalls by direct C–C addition).

Patented Applications of Gene Silencing in Plants: Manipulation of Traits and Phytopathogen Resistance

RNA silencing is the name of a broad family of phenomena including RNA interference (RNAi) in animals and basal eukaryotes, quelling in fungi and posttranscriptional gene silencing (PTGS) in plants. PTGS is a fertile research field and since its discovery many applications have been developed related to plant breeding. This minireview summarizes those patents which apply engineered gene silencing to specific problems. The range of inventions is divided in two main sections: manipulation of traits and resistance to phytopathogens and pests. Subtopics like manipulation of tolerances to abiotic stress, alteration of lignin, biofactories, alkaloids biosynthesis and flowering time fall within the first section, and introduction of resistances to insects, nematodes, bacteria, virus and fungi can be found within the second one.

Optimization of Agrobacterium-mediated transient gene expression and endogenous gene silencing in Piper colubrinum Link. by vacuum infiltration

The present study describes the successful development of vacuum infiltration method in the oomycete-resistant wild Piper sp., Piper colubrinum, as a rapid transient method for expression of GUS (β-Glucuronidase) reporter gene and introduction of hairpin vector for endogenous gene silencing. The GUS reporter gene construct pCAMBIA 1305.2 was used as a positive control to test the efficiency of vacuum infiltration strategy. Agrobacteria (EHA 105) harbouring GUS binary vector were vacuum-infiltrated into young detached in vitro leaf explants, which showed detectable GUS gene activity within 4 days of infiltration. This paper also reports for the first time the application of transient gene silencing in P. colubrinum by the delivery of in vitro synthesized hairpin vector construct (pHELLSGATE) containing endogenous serine threonine protein kinase (STPK) gene homologue into in vitro shoots. Introduction of hairpin vectors for the STPK gene into in vitro plantlets by vacuum infiltration resulted in significant reduction in transcript accumulation of the endogenous gene. The results indicate that transient gene silencing could be used as a rapid, preliminary high-throughput tool for P. colubrinum functional genomics.

PAMAM Dendrimers for siRNA Delivery: Computational and Experimental Insights

Short double-stranded RNAs, which are known as short interfering RNA (siRNA), can be used to specifically down-regulate the expression of the targeted gene in a process known as RNA interference (RNAi). However, the success of gene silencing applications based on the use of synthetic siRNA critically depends on efficient intracellular delivery. Polycationic branched macromolecules such as poly(amidoamine) (PAMAM) dendrimers show a strong binding affinity for RNA molecules and, hence, can provide an effective, reproducible, and relatively nontoxic method for transferring siRNAs into animal cells. Notwithstanding these perspectives, relatively few attempts have been made so far along these lines to study in detail the molecular mechanisms underlying the complexation process between PAMAMs and siRNAs. In this work we combine molecular simulation and experimental approaches to study the molecular requirements of the interaction of RNA-based therapeutics and PAMAM dendrimers of different generations. The dendrimers and their siRNA complexes were structurally characterized, and the free energy of binding between each dendrimer and a model siRNA was quantified by using the well-known MM/PBSA approach. DOSY NMR experiments confirmed the structural in silico prediction and yielded further information on both the complex structure and stoichiometry at low N/P ratio values. siRNA/PAMAM complex formation was monitored at different N/P ratios using gel retardation assays, and a simple model was proposed, which related the amount of siRNA complexed to the entropy variation upon complex formation obtained from the computer simulations.

RNA interference by nanofiber-based siRNA delivery system

SiRNA delivery has found useful applications particularly as therapeutic agents against genetic diseases. Currently, the delivery of siRNA typically takes the form of nanoparticles. In order to expand the applications of these potent but labile molecules for long-term use required by tissue engineering and regenerative medicine, alternative delivery vehicles are required. This work presents a scaffold-mediated approach to siRNA delivery. By encapsulating siRNA within polycaprolactone (PCL) nanofibers (300–400 nm in diameter) controlled release of intact siRNA could be achieved for at least 28 days under physiological conditions. The successful transfection of HEK 293 cells with GAPDH siRNA released from fibrous scaffolds at day 5, 15 and 30 demonstrated that the encapsulated molecules remained bioactive throughout the period of sustained release, providing silencing efficiency of 61–81% that was comparable to conventional siRNA transfection. Direct seeding of cells on these biofunctional scaffolds, with and without transfection reagent, demonstrated enhanced cellular uptake and efficient GAPDH gene-silencing. This work demonstrates the potential of nanofibrous scaffold-mediated siRNA delivery for long-term gene-silencing applications. The combination of topographical features provided by nanofibrous scaffolds may provide synergistic contact guidance and biochemical signals to mediate and support cellular development in regenerative medicine.

New Degradable Cationic Lipid-DNA Complexes for Gene Delivery

Cationic lipids (CLs) continue to attract attention as synthetic nucleic acid (NA) vectors, and are broadly used for gene transfection and silencing including applications in clinical trials. However, these complexes exhibit suboptimal gene expression due to inefficiencies in overcoming the cellular barriers to transfection (Ahmad et al., J. Gene Med. 2005, 7, 739). After entry of the CL-DNA complex into the cell, two major barriers are efficient dissociation of DNA from the complex and the cytotoxicity of CLs. To address both these issues, we have synthesized a novel series of multi¬valent lipids (CMVLs) with degradable disulfide bonds linking the positively charged headgroups of the CMVLs to their hydrophobic tails. The linker is designed to be cleaved in the reducing milieu of the cytoplasm thus facilitating CL-NA complex degradation, reducing cytotoxicity and improving NA release. X-ray scattering demonstrates that CMVLs form lamellar complexes with DNA, which degrade in reducing environments mimicking the cytoplasmic milieu. For lipids with highly charged headgroups such as CMVL5 (5+), X-ray diffraction under reducing conditions shows DNA condensed by the cleaved headgroup. No such condensation is observed for smaller headgroup charge, as in CMVL2 (2+). Most significantly, we observed an unexpectedly large reduction in cytotoxicity of degradable CMVL-vectors compared to vectors prepared from corresponding lipids without degradable bonds (MVLs). This is of particular importance for applications in gene silencing, where the delivery of short interfering RNAs (siRNAs) requires large CL/NA charge ratios. Correspondingly, the transfection efficiency of CMVL-DNA complexes remains high for high CL/DNA charge ratios. In summary, the much reduced cytotoxicity of these new degradable multivalent lipids and their propensity for DNA release in the cytoplasm open the way for the development of efficient non toxic CL-vectors of NAs. Funding provided by NIH GM-59288.

Biotechnology and Plant Disease Control-Role of RNA Interference

Development of crop varieties which are resistant against many economically important diseases is a major challenge for plant biotechnologists worldwide. Although much progress in this area has been achieved through classical genetic approaches, this goal can be achieved in a more selective and robust manner with the success of genetic engineering techniques. In this regard, RNA interference (RNAi) has emerged as a powerful modality for battling some of the most notoriously challenging diseases caused by viruses, fungi and bacteria. RNAi is a mechanism for RNA-guided regulation of gene expression in which double-stranded ribonucleic acid (dsRNA) inhibits the expression of genes with complementary nucleotide sequences. The application of tissue-specific or inducible gene silencing in combination with the use of appropriate promoters to silence several genes simultaneously will result in protection of crops against destructive pathogens. RNAi application has resulted in successful control of many economically important diseases in plants.

Applications of RNA interference-based gene silencing in animal agriculture

Classical genetic selection, recently aided by genomic selection tools, has been successful in achieving remarkable progress in livestock improvement. However, genetic selection has led to decreased genetic diversity and, in some cases, acquisition of undesirable traits. In order to meet the increased demands of our expanding population, new technologies and practices must be developed that contend with zoonotic and animal disease, environmental impacts of large farming operations and the increased food and fibre production needed to feed and clothe our society. Future increases in productivity may be dependent upon the acquisition of genetic traits not currently encoded by the genomes of animals used in standard agricultural practice, thus making classical genetic selection impossible. Genetic engineering of livestock is commonly used to produce pharmaceuticals or to impart enhanced production characteristics to animals, but has also demonstrated its usefulness in producing animals with disease resistance. However, significant challenges remain because it has been more difficult to produce animals in which specific genes have been removed. It is now possible to modify livestock genomes to block expression of endogenous and exogenous genes (such as those expressed following virus infection). In the present review, we discuss mechanisms of silencing gene expression via the biology of RNA interference (RNAi), the technology of activating the RNAi pathway and the application of this technology to enhance livestock production through increased production efficiency and prevention of disease. An increased demand for sustainable food production is at the forefront of scientific challenges and RNAi technology will undoubtedly play a key role.

Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

Efficient silencing of gene expression with modular trimeric Pol II expression cassettes comprising microRNA shuttles

Expressed polycistronic microRNA (miR) cassettes have useful properties that can be utilized for RNA interference (RNAi)-based gene silencing. To advance their application we generated modular trimeric anti-hepatitis B virus (HBV) Pol II cassettes encoding primary (pri)-miR-31-derived shuttles that target three different viral genome sites. A panel of six expression cassettes, comprising each of the possible ordering combinations of the pri-miR-31 shuttles, was initially tested. Effective silencing of individual target sequences was achieved in transfected cells and transcribed pri-miR trimers generated intended guide strands. There was, however, variation in processing and silencing by each of the shuttles. In some cases the monomers’ position within the trimers influenced processing and this correlated with target silencing. Compromised efficacy could be compensated by substituting the pri-miR-31 backbone with a pri-miR-30a scaffold. Inhibition of HBV replication was achieved in vivo, and in cell culture without disruption of endogenous miR function or induction of the interferon response. A mutant HBV target sequence, with changes in one of the guide cognates, was also silenced by the trimeric cassettes. The modular nature of the cassettes together with compatibility with expression from Pol II promoters should be advantageous for gene silencing applications requiring simultaneous targeting of different sites.

Therapeutic application of gene silencing MMP‐9 in a middle cerebral artery occlusion‐induced focal ischemia rat model

RNA interference appears to have a great potential not only as an in vitro target validation, but also as a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene. We hypothesize that MMP‐9 siRNA can be effective as an MMP‐9 protein inhibitor in a rat focal ischemia model. Male Sprague‐Dawley rats (156) were subjected to 2 h of middle cerebral artery occlusion (by using the suture insertion method)followed by 24 h of reperfusion. In the treatment group, 5 μl MMP‐9 siRNA was administrated by intracerebroventricular injection within 60 min after 2 h of focal ischemia. The siRNA transfection was demonstrated by fluorescence conjugated siRNA. Treatment with MMP‐9 siRNA produced a significant reduction in the cerebral infarction volume, brain water content, mortality rate and accompanying neurological deficits. The following were recorded: Evan's blue and IgG extravasation were reduced; the expression of MMP‐9 mRNA and protein were significantly silenced; and Immunohistochemistry and Western blot analysis revealed that the expression of MMP‐9 and VEGF were reduced while occludin and collagen‐ IV were up‐regulated in brain tissues. Our findings provide evidence that a liposomal formulation of siRNA might be used in vivo to silence the MMP‐9 gene and could potentially serve as an important therapeutic alternative in patients with cerebral ischemia.

Knocking down barriers: advances in siRNA delivery

In the 10 years that have passed since the Nobel prize-winning discovery of RNA interference (RNAi), billions of dollars have been invested in the therapeutic application of gene silencing in humans. Today, there are promising data from ongoing clinical trials for the treatment of age-related macular degeneration and respiratory syncytial virus. Despite these early successes, however, the widespread use of RNAi therapeutics for disease prevention and treatment requires the development of clinically suitable, safe and effective drug delivery vehicles. Here, we provide an update on the progress of RNAi therapeutics and highlight novel synthetic materials for the encapsulation and intracellular delivery of nucleic acids.

Functionalization of carbon nanotubes enables non-covalent binding and intracellular delivery of small interfering RNA for efficient knock-down of genes

The lipophilic nature of biological membranes restricts the direct intracellular delivery of potential drugs and molecular probes and makes intracellular transport one of the key problems in gene therapy. Because of their ability to cross cell membranes, single walled carbon nanotubes (SWNTs) are of interest as carriers of biologically active molecules, such as small interfering RNAs (siRNAs). We developed a strategy for chemical functionalization of SWNTs with hexamethylenediamine (HMDA) and poly(diallyldimethylammonium)chloride (PDDA) to obtain a material that was able to bind negatively charged siRNA by electrostatic interactions. PDDA–HMDA–SWNTs exhibited negligible cytotoxic effects on isolated rat heart cells at concentrations up to 10 mg/l. PDDA–HMDA–SWNTs loaded with extracellular signal-regulated kinase (ERK) siRNA were able to cross the cell membrane and to suppress expression of the ERK target proteins in primary cardiomyocytes by about 75%. PDDA-functionalized SWNTs thus present an effective carrier system for applications in siRNA-mediated gene silencing.

Liposomal Nanomedicines: An Emerging Field

Liposomal nanoparticles (LNs) encapsulating therapeutic agents, or liposomal nanomedicines (LNMs), represent one of the most advanced classes of drug delivery systems, with several currently on the market and many more in clinical trials. During the past 20 years, a variety of techniques have been developed for encapsulating both conventional drugs and the new genetic drugs (plasmid DNA-containing therapeutic genes, antisense oligonucleotides, and small, interfering RNA [siRNA]) within LNs encompassing a very specific set of properties: a diameter centered on 100 nm, a high drug-to-lipid ratio, excellent retention of the encapsulated drug, and a long (>6 hours) circulation lifetime. Particles with these properties tend to accumulate at sites of disease, such as tumors, where the endothelial layer is "leaky" and allows extravasation of particles with small diameters. Thus, LNs protect the drug during circulation, prevent it from reaching healthy tissues, and permit its accumulation at sites of disease. We will discuss recent advances in this field involving conventional anticancer drugs as well as gene-delivery, immunostimulatory, and gene-silencing applications involving the new genetic drugs. LNMs have the potential to offer new treatments in such areas as cancer therapy, vaccine development, and cholesterol management.

Reverse Genetics of Floral Scent: Application of Tobacco Rattle Virus-Based Gene Silencing in Petunia

Floral fragrance is responsible for attracting pollinators as well as repelling pathogens and pests. As such, it is of immense biological importance. Molecular dissection of the mechanisms underlying scent production would benefit from the use of model plant systems with big floral organs that generate an array of volatiles and that are amenable to methods of forward and reverse genetics. One candidate is petunia (Petunia hybrida), which has emerged as a convenient model system, and both RNAi and overexpression approaches using transgenes have been harnessed for the study of floral volatiles. Virus-induced gene silencing (VIGS) is characterized by a simple inoculation procedure and rapid results relative to transgenesis. Here, we demonstrate the applicability of the tobacco rattle virus-based VIGS system to studies of floral scent. Suppression of the anthocyanin pathway via chalcone synthase silencing was used as a reporter, allowing easy visual identification of anthocyaninless silenced flowers/tissues with no effect on the level of volatile emissions. Use of tobacco rattle virus constructs containing target genes involved in phenylpropanoid volatile production, fused to the chalcone synthase reporter, allowed simple identification of flowers with suppressed activity of the target genes. The applicability of VIGS was exemplified with genes encoding S-adenosyl-l-methionine:benzoic acid/salicylic acid carboxyl methyltransferase, phenylacetaldehyde synthase, and the myb transcription factor ODORANT1. Because this high-throughput reverse-genetics approach was applicable to both structural and regulatory genes responsible for volatile production, it is expected to be highly instrumental for large-scale scanning and functional characterization of novel scent genes.

Computational Estimation and Experimental Verification of Off-Target Silencing during Posttranscriptional Gene Silencing in Plants

Successful application of posttranscriptional gene silencing (PTGS) for gene function study in both plants and animals depends on high target specificity and silencing efficiency. By computational analysis with genome and/or transcriptome sequences of 25 plant species, we predicted that about 50% to 70% of gene transcripts in plants have potential off-targets when used for PTGS that could obscure experimental results. We have developed a publicly available Web-based computational tool called siRNA Scan to identify potential off-targets during PTGS. Some of the potential off-targets obtained from this tool were tested by measuring the amount of off-target transcripts using quantitative reverse transcription-PCR. Up to 50% of the predicted off-target genes tested in plants were actually silenced when tested experimentally. Our results suggest that a high risk of off-target gene silencing exists during PTGS in plants. Our siRNA Scan tool is useful to design better constructs for PTGS by minimizing off-target gene silencing in both plants and animals.

Conjugates of Oligonucleotides and Analogues with Cell Penetrating Peptides as Gene Silencing Agents

The review describes key aspects of the synthesis and biological activities of conjugates of oligonucleotides and their analogues with synthetic peptides, in particular aimed towards gene silencing applications. The common methods of synthesis of oligonucleotide-peptide conjugates (OPCs) and PNA-peptide conjugates (PPCs) are described, which include both total solid-phase and fragment coupling approaches. In addition, various applications of conjugates as gene silencing agents are outlined. These include antisense and steric block applications in mammalian cells of OPCs, PPCs and phosphorodiamidate morpholinooligonucleotide (PMO)-peptide conjugates, gene silencing in bacteria, various DNA targeting applications, and recent reports of gene silencing activities of siRNA-peptide conjugates. A table listing all peptides used as oligonucleotide conjugates for gene silencing applications is also included.

Small interfering RNA delivery and gene silencing using polymeric nanocarrier systems

The effectiveness of a drug is determined by the ability to migrate through the body and reach target sites in therapeutically relevant levels. Nanocarriers for delivery of bioactive agents are being developed at iNANO to maximise drug payload at target sites. The inclusion of “biological triggers” into the nanocarrier design is used for modulation of cellular nucleic acid trafficking and increased target interaction.Polymers were used to formulate nanocarriers in the size range 30–250 nm containing small interfering RNAs (siRNAs) for gene silencing applications. PAGE analysis showed the structural integrity of the siRNA was maintained during particle formation. In systems composed of bioresponsive polymers, nanocarrier disassembly and siRNA release under cellular conditions were shown, using Atomic Force Microscopy. The time course for siRNA uptake into NIH cells was visualised using confocal microscopy. In addition, siRNA localisation within cells could be modulated by the composition of the polymer used. The ability of the nanocarrier system to mediate gene expression was investigated in a cell line stably expressing enhanced green fluorescent protein (eGFP). Furthermore, the various delivery systems were tested in a mouse model stably expressing the eGFP protein using both nasal and intravenous delivery routes.The systems described in this work demonstrate an ability to transport siRNA into cells whilst maintaining siRNA functionality; essential properties for nanocarrier-based RNA interference strategies.

RNA hydrolysis and inhibition of translation by a Co(III)–cyclen complex

Metal ion-chelator catalysts based on main-group, lanthanide, or transition metal complexes have been developed as nonenzymatic alternatives for the hydrolysis of the phosphodiester bonds in DNA and RNA. Cobalt (III), with its high-charge density, is known for its ability to hydrolyze phosphodiesters with rate constants as high as 2 x 10(-4) s(-1). We have developed a kinetically inert Co(III)-cyclen-based complex, Co(III)-cycmmb that is very potent in inhibiting the translation of RNA into protein. Contact time as short as 10 min is sufficient to achieve the complete inhibition of the translation of a concentrated luciferase RNA solution into the enzyme in a cell-free translation system. The inhibition appears to proceed through two pathways. The first pathway involves the kinetic or substitutional inertness of Co(III) for the RNA template at short contact times. This interaction is mediated through the kinetic inertness of Co(III) for the phosphate groups of the nucleotides, as well as coordination of Co(III) to the nitrogenous bases. The second pathway occurs at longer contact times and is mediated by the hydrolysis of the phosphodiester backbone. This report represents the first demonstrated use of a metal-chelate complex to achieve the inhibition of the translation of RNA into protein. This Co(III) system can be useful in its present nonsequence-specific form as a novel viral decontamination agent. When functionalized to recognize specific nucleic acid sequences, such a system could potentially be used in gene-silencing applications as an alternative to standard antisense or RNAi technologies.