siRNA Gene Silencing Research Articles

Enhancing host defense against bacterial pore-forming toxin by suppressing HMG-CoA reductase pathway in airway epithelial cells (112.13)

Abstract Pneumolysin and α-hemolysin are the main pore-forming toxins known to play critical roles in bacterial pneumonia caused by the infections of Streptococcus pneumoniae and Staphylococcus aureus, respectively. Airway epithelia are the initial and the primary targets of these air-borne bacterial infections. Several epidemiological studies have linked the use of statin to the improvement in pneumonia outcomes, but the nature of the mechanism is still unknown. Using primary normal human bronchial epithelial (NHBE) cells and an immortalized normal bronchial epithelial cell line, HBE1, we confirmed the effects of simvastatin pre-treatment in enhancing host defense against these bacterial toxins. The protective effects could be further demonstrated in vivo with simvastatin administration prior to intra-tracheal instillation of these toxins. We further found that the protection requires protein synthesis and is calcium dependent. Using siRNA gene silencing, pharmacological treatment with inhibitors, immuno fluorescence microscopy, Western blot and qRT-PCR approaches, we have delineated the protection mechanisms in airway epithelial cells through HMG-CoA-Reductase pathways. Our results also suggest that the inhibition of HMG-CoA reductase pathway may enhance host defense molecule synthesis in airway epithelium. It is plausible that this study may lead to the development of an adjuvant therapy against pore-forming toxin-related bacterial infections in lung.

Potent Gene Silencing In Vitro at Physiological pH Using Chitosan Polymers

Among non-viral cationic polymers, biodegradable chitosan has during the last decade become an attractive carrier for small interference RNA (siRNA) delivery. Currently, degradation of macromolecules in the lysosomes is assumed to be a major barrier for effective siRNA transfection. Hence, transfection protocols are focused toward endosomal release mechanisms. In this work, we have tested 3 novel chitosan polymers and their siRNA delivery properties in vitro. To obtain efficient gene silencing of our model gene, S100A4, various transfection parameters were investigated, such as pH, nitrogen/phosphate ratio, photochemical internalization (PCI), media for complex formation, and cell lines. Our results showed that 2 linear chitosan polymers demonstrated excellent siRNA gene silencing, better than Lipofectamine 2000. The silencing effect was achieved without PCI treatment, under physiological pH, and with no observable reduction in cell viability.

Mouldy Sioud (ed): siRNA and miRNA Gene Silencing: from Bench to Bedside

This is one of the most comprehensive and recent books about gene targeting through siRNA and miRNA, intended to provide readers with an updated compendium of current strategies and methods towards therapeutic applications of gene silencing. It starts with topics related to the optimization of siRNAs and their performance in vivo, mentioning design guide rules and new methods useful for designing siRNA sequences (i.e., statistical and clustering techniques) as well as chemical modifications (i.e., LNA) to prevent unwanted effects or activation of innate immunity. Nevertheless, the main challenges in using siRNA in vivo are the delivery, tissue/cell specific targeting and monitoring of siRNA release and potency in vivo. Therefore, most chapters are dedicated to these issues, focusing on several methods for siRNA delivery in live animals, such as magnetofection or nanoparticles using peptide–receptor interaction or atelocollagen matrix, and imaging approaches to monitor in vivo siRNA release and its effects (bioluminescence, MRI, radiolabelling). The book also covers topics related to intriguing new technologies, such as the use of TransKingdom RNAi (tkRNAi, Chapter 7) and bacterial mediated delivery of siRNA in solid tumors (Chapter 8). Special attention is given to therapeutic application of gene silencing through miRNA and siRNA, mentioning relevant examples of therapeutic target genes already validated in vitro or in vivo (Chapters 11–16), including oncogenes and viral proteins. Indeed, the book ends by describing the use of a synthetic siRNA against Bcr–Abl fusion transcript in a patient with leukemia. While being very detailed in the issues concerning siRNAs and their application in vivo, only few chapters are dedicated to miRNAs, mainly describing the microRNA pathway and biogenesis, with few mentions on miRNA therapeutics. The text is suited for researchers, teachers, students and biotech companies interested in siRNAand miRNA-based approach to gene regulation, which could benefit of a recently updated and comprehensive compendium of methods and strategies envisioned to foster the development of RNAi technology from bench to bedside.

SiRna Transfection and Gene Silencing in Human Aortic Smooth Muscle Cells from Electrospun PET

SiRna Transfection and Gene Silencing in Human Aortic Smooth Muscle Cells from Electrospun PET

Biodegradable Nanoparticles of mPEG-PLGA-PLL Triblock Copolymers as Novel Non-Viral Vectors for Improving siRNA Delivery and Gene Silencing

Degradation of mRNA by RNA interference is one of the most powerful and specific mechanisms for gene silencing. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. Various physicochemical properties of mPEG-PLGA-PLL NPs, including morphology, size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by scanning electron microscope, particle size and zeta potential analyzer, and high performance liquid chromatography. The levels of siRNA uptake and targeted gene inhibition were detected in human lung cancer SPC-A1-GFP cells stably expressing green fluorescent protein. Examination of the cultured SPC-A1-GFP cells with fluorescent microscope and flow cytometry showed NPs loading Cy3-labeled siRNA had much higher intracellular siRNA delivery efficiencies than siRNA alone and Lipofectamine-siRNA complexes. The gene silencing efficiency of mPEG-PLGA-PLL NPs was higher than that of commercially available transfecting agent Lipofectamine while showing no cytotoxicity. Thus, the current study demonstrates that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing.

Complexing a small interfering RNA with divalent cationic surfactants

Small interfering RNAs (siRNAs) are double strand RNA fragments of short sequence (∼20 bp). RNA interference came into focus only 13 years ago as a major biological breakthrough and, since then, many studies have described the involvement of siRNA in gene silencing. Application to gene therapy is extremely promising, provided that appropriate vectors are used. Optimising transfection efficacy strongly relies on the knowledge and tuning of physicochemical properties of transfection complexes, such as size, surface charge and internal interactions, which govern in vitro and in vivo stability. Here we report a study on siRNA complexation with micelles of two types of divalent cationic surfactants, i.e. three Gemini bis(quaternary ammonium) bromide with variable spacer length (12-3-12, 12-6-12, 12-12-12) and one weak electrolyte surfactant with a triazine polar head. The process of complex formation was followed by SANS, DLS and zeta potential. Charge density on micelles and counterion exchange were key factors in determining the extent of complexation, as it happens to polymer electrolytes interacting with micelles. A description of complex formation was given in terms of liquid–liquid micro-phase separation, due to internally structured coacervates progressively nucleating from the micelle solution upon siRNA addition. An affinity order between surfactants and siRNA could be established on the basis of the obtained results and their comparison.

Matrilin-3 Induction of IL-1 receptor antagonist Is required for up-regulating collagen II and aggrecan and down-regulating ADAMTS-5 gene expression

IntroductionDeletion or mutation of the gene encoding the cartilage extracellular matrix (ECM) protein matrilin-3 (MATN3) results in the early onset of osteoarthritis (OA), suggesting chondroprotective properties of MATN3. To understand the mechanisms underlying these properties, we determined the effects of MATN3 protein on the expression of several key anabolic and catabolic genes involved in chondrocyte homeostasis, and the dependence of such regulation on the anti-inflammatory cytokine: IL-1 receptor antagonist (IL-1Ra).MethodsThe effects of recombinant human (rh) MATN3 protein were examined in C28/I2 immortalized human chondrocytes, primary human chondrocytes (PHCs), and primary mouse chondrocytes (PMCs). Messenger RNA levels of IL-1Ra, COL2A1, ACAN, MMP-13, and ADAMTS-4 and -5 were determined using real-time RT-PCR. Knocking down IL-1Ra was achieved by siRNA gene silencing. IL-1Ra protein levels were quantified by ELISA and the Bio-Plex Suspension Array System. COL2A1 protein level was quantified using Western blot analysis. Statistic analysis was done using the two-tailed t-test or one-way ANOVA.ResultsrhMATN3 protein induced gene expression of IL-1Ra in C28/I2 cells, PHCs, and PMCs in a dose- and time-dependent manner. Treatment of C28/I2 cells and PHCs with MATN3 protein stimulated gene expression of COL2A1 and ACAN. Conversely, mRNA levels of COL2A1 and ACAN were decreased in MATN3 KO mice. MATN3 protein treatment inhibited IL-1β-induced MMP-13, ADAMTS-4 and ADAMTS-5 in C28/I2 cells and PHCs. Knocking down IL-1Ra abolished the MATN3-mediated stimulation of COL2A1 and ACAN and inhibition of ADAMTS-5, but had no effect on MATN3 inhibition of MMP-13 mRNA.ConclusionOur findings point to a novel regulatory role of MATN3 in cartilage homeostasis due to its capacity to induce IL-1Ra, to upregulate gene expression of the major cartilage matrix components, and to downregulate the expression of OA-associated matrix-degrading proteinases in chondrocytes. The chondroprotective properties of endogenous MATN3 depend partly on its induction of IL-1Ra. Our findings raise a possibility to use rhMATN3 protein for anti-inflammatory and chondroprotective therapy.

Comparison of Cationic and Amphipathic Cell Penetrating Peptides for siRNA Delivery and Efficacy

Cell penetrating peptides (CPPs) are short strands of arginine- and/or lysine-rich peptides (<30 amino acids) that use their cationic nature for efficient intracellular accumulation. CPPs have been used for small interfering RNA (siRNA) delivery by direct complexation with the siRNA anionic phosphate backbone. During this process, however, part of the CPP cationic charges are neutralized, and the resultant loss of free positive charges may substantially compromise CPP's internalization capabilities and eventually reduce siRNA delivery efficiency. The purpose of this study was to design a novel type of polyplex for siRNA delivery to overcome the CPP neutralization issue. This novel polyplex consists of three components: siRNA, 21mer oligolysine (K21) chemically modified to incorporate CPP conjugation sites (K21-PDP), and CPP delivery moiety. The siRNA was first neutralized by cationic charges of K21-PDP to form a polyplex. Then a cationic (hexaarginine, R6) or an amphipathic (model amphipathic peptide, MAP) CPP was conjugated to the polyplex. Agarose gel shift assays indicated that the siRNA could be released from the polyplex after K21-PDP degradation or polyplex dilution. Furthermore, the total intracellular internalization of these two CPP-polyplexes was studied. Compared with R6-polyplex, MAP-polyplex exhibited 170- and 600-fold greater uptake of fluorescently labeled siRNA at 1 and 6 h post-transfection, respectively. MAP-polyplex also exhibited comparable GFP silencing effects as Lipofectamine 2000 complex in Huh7.5 cells stably transfected to express GFP-light chain 3 protein, whereas R6-polyplex did not demonstrate significant silencing activity. Further studies indicated that the K21-PDP-siRNA polyplex formation and conjugation of MAP to the polyplex were essential for siRNA polyplex uptake and gene silencing. MAP-polyplex was also shown to be unaffected by the presence of 10% FBS during transfection. In addition, MAP-polyplex uptake was dependent on vesicle formation and fusion due to 70 and 54% loss of uptake at 4 and 16 °C, respectively, compared to incubation at 37 °C. Therefore, the amphipathic CPP is a more suitable carrier moiety for delivery of siRNA polyplex.

Sec62 Bridges the Gap from 3q Amplification to Molecular Cell Biology in Non–Small Cell Lung Cancer

The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non-small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro. Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62-overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non-small cell lung cancer and harbors various functions in cancer cell biology.

NAV3 copy number changes and target genes in basal and squamous cell cancers

The neuron navigator 3 (NAV3) gene on chromosome 12q21 encodes a microtubule plus end tracking protein and belongs to the navigator family of cytoskeletal regulators. Loss of heterozygosity on 12q has previously been suggested to be associated with poor prognosis in cancers of epithelial origin. In this study, we characterized copy number changes of NAV3 in 24 basal cell cancers (BCCs), eight squamous cell cancers (SCCs) and eight non-malignant inflammatory skin lesions by fluorescent in situ hybridization. To identify genes affected by NAV3, we used oligo siRNA gene silencing and gene microarrays to analyse gene expression profiles at several time points post-transfection in primary human keratinocytes. We found NAV3 copy number loss and decreased protein expression in 21% of the BCCs and 25% of the SCCs. In the nodular/superficial BCC subgroup, low-level NAV3 amplification was also observed. NAV3 aberrations were independent of the known chromosome 6 amplifications in BCC. Chromosome 12 polysomy was detected in 33% and 25% of the invasive type of BCC and SCC, respectively. Silencing of NAV3 in primary human keratinocytes revealed 22 differentially expressed genes, mostly related to inflammation. The most relevant of these were validated with qPCR or immunohistochemistry. This pilot study suggests that NAV3 is a novel cancer-associated gene that contributes to the pathogenesis of a subgroup of BCC and SCC.

Modified siRNA Structure With a Single Nucleotide Bulge Overcomes Conventional siRNA-mediated Off-target Silencing

Off-target gene silencing is a major concern when using RNA interference. Imperfect pairing of the antisense strand with unintended mRNA targets is one of the main causes of small interfering RNA (siRNA) off-target silencing. To overcome this, we have developed "bulge-siRNA," a modified siRNA backbone structure with a single nucleotide (nt) bulge placed in the antisense strand. We found that siRNAs with a bulge at position 2 of the antisense strand were able to discriminate better between perfectly matched and mismatched targets, with no loss in silencing of the intended target. Genome-wide analysis also revealed that the bulge-siRNAs significantly reduced off-target silencing of transcripts with complementarity to the seed region of the siRNA antisense strand. When compared to 2'-methoxy ribosyl (2'-OMe) modified siRNAs previously developed to alleviate antisense off-target silencing; the bulge modification could better discriminate between on- versus off-targets. Our results suggest that the bulge-siRNA structure is a simple, yet superior alternative to chemical modifications for minimizing off-target silencing triggered by conventional siRNA structures.

Effects of RNA interference-induced Caveolin-1 downregulation on lung edema of rats

Objective To explore the effects of down-regulation of Caveolin-1 gene expression on the permeability of lung capillaries. Methods Small interfering RNA (siRNA) was used to knock-down Caveolin-1 expression in rat lungs. A single-stranded siRNA construct was designed, and another sequence was used as a control construct. Pulmonary injection of siRNA against Caveolin-1 mRNA incorporated in liposomes with three dosage gradients of 0. 4, 0. 8 and 1.2 mg/kg. The protein expression of Caveolin-1 was detected by Western blotting and immunohistochemistry. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated the expression of caveolin-1 expression. The expression of Aquaporin (AQP)-1protein was detected by using Western blotting. Chest fluoroscopy showed the degree of lung edema. 1% of Evans blue was injected through vena caudalis with a dosage of 1.0 mL/kg, and then lobus medius pulmonis was dissected and immersed in formamide. Results At 72nd h in experimental group, the gene silencing of Caveolin-1 siRNA was about 85%. There was slight difference in the Caveolin-1 expression in the control group with the three dosage gradients. Caveolin-1 siRNA had little influence over the AQP-1 protein level. Under the chest fluoroscopy, density of pulmonary lobes was increased. In comparison with the control group, the absorbance values of formamide in experimental group were significantly increased (P ＜0. 01 ). Conclusion Caveolin-1 may play an important role in the process of lung edema, which was AQP-1 independent. Key words: Lung; Caveolin-1; Permeability; Aquaporin

Multifunctional quantum-dot-based siRNA delivery for HPV18 E6 gene silence and intracellular imaging

The functional quantum dots (QDs) were specifically designed to overcome barriers in siRNA delivery such as siRNA protection, cellular penetration, endosomal release, carrier unpacking, intracellular transport and gene silencing. In this paper, two l-arginine-functional-modified CdSe/ZnSe QDs were synthesized as siRNA carriers to silence HPV18 E6 gene in HeLa cells. Using such constructs, these QDs showed significantly low cellular cytotoxicity and good siRNA protection. Flow cytometric and confocal microscopic analyses confirmed that the QDs delivered siRNA into HeLa cells efficiently. Importantly, superior gene silencing efficiency was achieved as evaluated by Reverse Transcription-PCR (RT-PCR) and Western blotting and HeLa cells growth was inhibited in xCELLigence installation analysis and MTT assay when treated with QD-siRNA complexes. Interestingly, the QDs coated with β-CD- l-Arg showed optimized property compared with those coated with l-Arg. Furthermore, these QDs complexes could also be used as nanocrystal probing agents, allowing real-time tracking and localization of QDs during delivery and transfection. The properties and capabilities of these QDs showed that amino acid-modified QDs could be used as useful siRNA carriers to effectively silence a target gene as well as fluorescence probes to analyze intracellular imaging in vivo.

Endogenous erythropoietin from astrocyte protects the oligodendrocyte precursor cell against hypoxic and reoxygenation injury

The hypoxia-responsive cytokine erythropoietin (EPO) provides neuroprotective effects in the damaged brain during ischemic events and neurodegenerative diseases. The purpose of the present study is to evaluate the EPO/EPO receptor (EPOR) endogenous system between astrocyte and oligodendrocyte precursor cell (OPC) under hypoxia. We report here elevated EPO mRNA levels and protein release in cultured astrocytes following hypoxic stimulation by quantitative RT-PCR and ELISA. Furthermore, the EPOR gene expressions were detected in cultured OPCs as in astrocytes and microglias by quantitative RT-PCR. Cell staining revealed the EPOR expression in OPC. To evaluate the protective effect of endogenous EPO from astrocyte to OPCs, EPO/EPOR signaling was blocked by EPO siRNA or EPOR siRNA gene silencing in in vitro study. The suppression of endogenous EPO production in astrocytes by EPO siRNA decreased the protection to OPCs against hypoxic stress. Furthermore, OPC with EPOR siRNA had less cell survival after hypoxic/reoxygenation injury. This suggested that EPO/EPOR signaling from astrocyte to OPC could prevent OPC damage under hypoxic/reoxygenation condition. Our present finding of an interaction between astrocytes and OPCs may lead to a new therapeutic approach to OPCs for use against cellular stress and injury.

Functional motor recovery from brain ischemic insult by carbon nanotube-mediated siRNA silencing.

Stroke is the second cause of death worldwide with ischemic stroke accounting for 80% of all stroke insults. Caspase-3 activation contributes to brain tissue loss and downstream biochemical events that lead to programmed cell death after traumatic brain injury. Alleviation of symptoms following ischemic neuronal injury can be potentially achieved by either genetic disruption or pharmacological inhibition of caspases. Here, we studied whether silencing of Caspase-3 using carbon nanotube-mediated in vivo RNA interference (RNAi) could offer a therapeutic opportunity against stroke. Effective delivery of siRNA directly to the CNS has been shown to normalize phenotypes in animal models of several neurological diseases. It is shown here that peri-lesional stereotactic administration of a Caspase-3 siRNA (siCas 3) delivered by functionalized carbon nanotubes (f-CNT) reduced neurodegeneration and promoted functional preservation before and after focal ischemic damage of the rodent motor cortex using an endothelin-1 induced stroke model. These observations illustrate the opportunity offered by carbon nanotube-mediated siRNA delivery and gene silencing of neuronal tissue applicable to a variety of different neuropathological conditions where intervention at well localized brain foci may offer therapeutic and functional benefits.

Introducing the Korean Oligonucleotide Therapeutics Society

Nucleic Acid TherapeuticsVol. 21, No. 3 Articles from the Korean Oligonucleotide Therapeutics Society (K-OTS)Guest EditorialIntroducing the Korean Oligonucleotide Therapeutics SocietySeong-Wook LeeSeong-Wook LeeSearch for more papers by this authorPublished Online:29 Jun 2011https://doi.org/10.1089/nat.2011.1500AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Introducing the Korean Oligonucleotide Therapeutics Society." , 21(3), p. 123FiguresReferencesRelatedDetails Volume 21Issue 3Jun 2011 InformationCopyright 2011, Mary Ann Liebert, Inc.To cite this article:Seong-Wook Lee.Introducing the Korean Oligonucleotide Therapeutics Society.Nucleic Acid Therapeutics.Jun 2011.123-123.http://doi.org/10.1089/nat.2011.1500Published in Volume: 21 Issue 3: June 29, 2011PDF download

Structural Diversity Repertoire of Gene Silencing Small Interfering RNAs

Since the discovery of double-stranded (ds) RNA-mediated RNA interference (RNAi) phenomenon in Caenorhabditis elegans, specific gene silencing based upon RNAi mechanism has become a novel biomedical tool that has extended our understanding of cell biology and opened the door to an innovative class of therapeutic agents. To silence genes in mammalian cells, short dsRNA referred to as small interfering RNA (siRNA) is used as an RNAi trigger to avoid nonspecific interferon responses induced by long dsRNAs. An early structure-activity relationship study performed in Drosophila melanogaster embryonic extract suggested the existence of strict siRNA structural design rules to achieve optimal gene silencing. These rules include the presence of a 3' overhang, a fixed duplex length, and structural symmetry, which defined the structure of a classical siRNA. However, several recent studies performed in mammalian cells have hinted that the gene silencing siRNA structure could be much more flexible than that originally proposed. Moreover, many of the nonclassical siRNA structural variants reported improved features over the classical siRNAs, including increased potency, reduced nonspecific responses, and enhanced cellular delivery. In this review, we summarize the recent progress in the development of gene silencing siRNA structural variants and discuss these in light of the flexibility of the RNAi machinery in mammalian cells.

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

Multifunctional Triblock Nanocarrier (PAMAM-PEG-PLL) for the Efficient Intracellular siRNA Delivery and Gene Silencing

A novel triblock poly(amido amine)-poly(ethylene glycol)-poly-l-lysine (PAMAM-PEG-PLL) nanocarrier was designed, synthesized, and evaluated for the delivery of siRNA. The design of the nanocarrier is unique and provides a solution to most of the common problems associated with the delivery and therapeutic applications of siRNA. Every component in the triblock nanocarrier plays a significant role and performs multiple functions: (1) tertiary amine groups in the PAMAM dendrimer work as a proton sponge and play a vital role in the endosomal escape and cytoplasmic delivery of siRNA; (2) PEG, a linker connecting PLL and PAMAM dendrimers renders nuclease stability and protects siRNA in human plasma; (3) PLL provides primary amines to form polyplexes with siRNA through electrostatic interaction and also acts as penetration enhancer; and (4) conjugation to PEG and PAMAM reduced toxicity of PLL and the entire triblock nanocarrier PAMAM-PEG-PLL. The data obtained show that the polyplexes resulted from the conjugation of siRNA, and the proposed nanocarriers were effectively taken up by cancer cells and induced the knock down of the target BCL2 gene. In addition, triblock nanocarrier/siRNA polyplexes showed excellent stability in human plasma.

Modeling RNA interference in mammalian cells

BackgroundRNA interference (RNAi) is a regulatory cellular process that controls post-transcriptional gene silencing. During RNAi double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous mRNA via the generation of smaller dsRNA oligomers of length between 21-23nt (siRNAs). siRNAs are then loaded onto the RNA-Induced Silencing multiprotein Complex (RISC), which uses the siRNA antisense strand to specifically recognize mRNA species which exhibit a complementary sequence. Once the siRNA loaded-RISC binds the target mRNA, the mRNA is cleaved and degraded, and the siRNA loaded-RISC can degrade additional mRNA molecules. Despite the widespread use of siRNAs for gene silencing, and the importance of dosage for its efficiency and to avoid off target effects, none of the numerous mathematical models proposed in literature was validated to quantitatively capture the effects of RNAi on the target mRNA degradation for different concentrations of siRNAs. Here, we address this pressing open problem performing in vitro experiments of RNAi in mammalian cells and testing and comparing different mathematical models fitting experimental data to in-silico generated data. We performed in vitro experiments in human and hamster cell lines constitutively expressing respectively EGFP protein or tTA protein, measuring both mRNA levels, by quantitative Real-Time PCR, and protein levels, by FACS analysis, for a large range of concentrations of siRNA oligomers.ResultsWe tested and validated four different mathematical models of RNA interference by quantitatively fitting models' parameters to best capture the in vitro experimental data. We show that a simple Hill kinetic model is the most efficient way to model RNA interference. Our experimental and modeling findings clearly show that the RNAi-mediated degradation of mRNA is subject to saturation effects.ConclusionsOur model has a simple mathematical form, amenable to analytical investigations and a small set of parameters with an intuitive physical meaning, that makes it a unique and reliable mathematical tool. The findings here presented will be a useful instrument for better understanding RNAi biology and as modelling tool in Systems and Synthetic Biology.