A structural interpretation of the effect of GC-content on efficiency of RNA interference

BACKGROUND: RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful technique for eukaryotic gene knockdown. siRNA GC-content negatively correlates with RNAi efficiency, and it is of interest to have a convincing mechanistic interpretation of this observation. We here examine this issue by considering the secondary structures for both the target messenger RNA (mRNA) and the siRNA guide strand. RESULTS: By analyzing a unique homogeneous data set of 101 shRNAs targeted to 100 endogenous human genes, we find that: 1) target site accessibility is more important than GC-content for efficient RNAi; 2) there is an appreciable negative correlation between GC-content and RNAi activity; 3) for the predicted structure of the siRNA guide strand, there is a lack of correlation between RNAi activity and either the stability or the number of free dangling nucleotides at an end of the structure; 4) there is a high correlation between target site accessibility and GC-content. For a set of representative structural RNAs, the GC content of 62.6% for paired bases is significantly higher than the GC content of 38.7% for unpaired bases. Thus, for a structured RNA, a region with higher GC content is likely to have more stable secondary structure. Furthermore, by partial correlation analysis, the correlation for GC-content is almost completely diminished, when the effect of target accessibility is controlled. CONCLUSION: These findings provide a target-structure-based interpretation and mechanistic insight for the effect of GC-content on RNAi efficiency

A Structural Interpretation of the Effect of GC-Content on Efficiency of RNA Interference

BACKGROUND: RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful technique for eukaryotic gene knockdown. siRNA GC-content negatively correlates with RNAi efficiency, and it is of interest to have a convincing mechanistic interpretation of this observation. We here examine this issue by considering the secondary structures for both the target messenger RNA (mRNA) and the siRNA guide strand. RESULTS: By analyzing a unique homogeneous data set of 101 shRNAs targeted to 100 endogenous human genes, we find that: 1) target site accessibility is more important than GC-content for efficient RNAi; 2) there is an appreciable negative correlation between GC-content and RNAi activity; 3) for the predicted structure of the siRNA guide strand, there is a lack of correlation between RNAi activity and either the stability or the number of free dangling nucleotides at an end of the structure; 4) there is a high correlation between target site accessibility and GC-content. For a set of representative structural RNAs, the GC content of 62.6% for paired bases is significantly higher than the GC content of 38.7% for unpaired bases. Thus, for a structured RNA, a region with higher GC content is likely to have more stable secondary structure. Furthermore, by partial correlation analysis, the correlation for GC-content is almost completely diminished, when the effect of target accessibility is controlled. CONCLUSION: These findings provide a target-structure-based interpretation and mechanistic insight for the effect of GC-content on RNAi efficiency

Design of RNAi reagents for invertebrate model organisms and human disease vectors

RNAi has become an important tool to silence gene expression in a variety of organisms, in particular when classical genetic methods are missing. However, application of this method in functional studies has raised new challenges in the design of RNAi reagents in order to minimize false positive and false negative results. Since the performance of reagents can be rarely validated on a genome-wide scale, improved computational methods are required that consider experimentally derived design parameters. Here, we describe computational methods for the design of RNAi reagents for invertebrate model organisms and human disease vectors, such as Anopheles. We describe procedures on how to design short and long double-stranded RNAs for single genes, and evaluate their predicted specificity and efficiency. Using a bioinformatics pipeline we also describe how to design a genome-wide RNAi library for Anopheles gambiae

Development of a bioactive coating for the specific gene silencing of pathogenetic processes after intravascular stent implantation

Kardiovaskuläre Erkrankungen weisen die höchste Sterblichkeitsrate in der westlichen Welt auf. Zu ihnen gehören viele verschiedene Krankheiten, wie z.B. die koronare Herzkrankheit (KHK). Der Hauptauslöser für die Entwicklung dieser Erkrankungen ist die Atherosklerose. Sie ist ein fortschreitender, entzündlicher Prozess, bei dem eine Verengung der Arterien durch die Bildung von atherosklerotischen Plaques auftritt. Dadurch kann weniger Blut zum Herzen transportiert werden, was zu einer generellen Unterversorgung des Herzmuskels oder im schlimmsten Fall zu einem Myokardinfarkt führen kann. Um den natürlichen Blutfluss in dem verengten Abschnitt der Arterie wiederherzustellen, sind zwei interventionelle Therapieoptionen üblich: 1) die Koronararterien-Bypass-Operation (CABG) oder 2) die perkutane transluminale Koronarangioplastie (PTCA). Während bei der CABG überwiegend autologe Venentransplantate verwendet werden, wird bei der PTCA ein Ballon für die Erweiterung der Arterie verwendet, mit der Option, einen Stent über den Katheter zu platzieren. Beim Einbringen der bisher üblichen Stents, wie Bare Metal Stents (BMS) oder Drug Eluting Stents (DES), kann das Endothel durch das Expandieren des Stents und durch dessen Drahtgeflecht geschädigt werden und es kann zu einer nachfolgenden Entzündungsreaktion und einem Wiederverschluss (Restenose) kommen. Mit den DES wird dieses Risiko minimiert, da sie antiproliferative oder immunsuppressive Wirkstoffe freisetzen. Jedoch birgt dieser Vorteil auch einen Nachteil, da die Wirkstoffe gleichzeitig das Wachstum von Endothelzellen hemmen und Patienten länger auf antithrombotische Medikamente angewiesen sind. Gegenwärtig gibt es viele verschiedene Ansätze zur Entwicklung von Stents der nächsten Generation, um unerwünschte Reaktionen wie die In-Stent-Restenose (ISR) und eine späte Stentthrombose zu verringern. Ein initialer Prozess während der Entwicklung der Atherosklerose und nach Verletzung des arteriellen Gewebes durch die Stent-Implantation ist die Rekrutierung von Leukozyten. Diese wird durch verschiedene zelluläre Adhäsionsmoleküle (CAMs) wie z. B. dem interzellulären Adhäsionsmolekül 1 (ICAM–1), welches im Bereich der Verletzung von Endothelzellen vermehrt gebildet wird, vermittelt. ICAM–1 gehört zur sogenannten Leukozytenadhäsionsmolekülkaskade. Wenn es möglich wäre die Bildung des ICAM–1 sowie die Bildung weiterer relevanter Proteine spezifisch durch siRNA zu hemmen, so könnte die initiale Leukozytenadhäsionsmolekülkaskade durchbrochen und die Restenosegefahr vermindert werden. Drei verschiedene Stent-Beschichtungssysteme zur substratvermittelten small interfering RNA (siRNA)-Freisetzung gegen ICAM–1 wurden in dieser Arbeit entwickelt. Als Basis für die Schichtsysteme dienten Lipoplexe aus Lipofectamine®2000 und spezifische sowie fluoreszenzmarkierte siRNA oder Polyplexe aus Poly(ethylenimin) (PEI) und siRNA. Als Biomaterialien für die Einbettung der Lipo- oder Polyplexe wurden Atelokollagen, verschiedene Poly(lactid-co-glycolid) (PLGA) Resomere oder Polyelektrolyt-Mehrschicht (PEM)-Schichtsysteme aus PEI und Hyaluronsäure (HA) verwendet. Die Aufnahme bzw. Transfektion von siRNA in Zellen der Zelllinie EA.hy926 konnte mit allen drei Stent-Beschichtungssystemen nachgewiesen werden. Darüber hinaus wurde untersucht, ob die Schichtsysteme über eine Langzeitfreisetzung verfügen oder die siRNA über einen kurzen Zeitraum abgeben. Die langfristige Freisetzung von siRNA ist besonders interessant für die Stent-Therapie, da der Stent in der Arterie verbleibt, um ihn offen zu halten, und die Heilung eines verletzten Endothels bis zu drei Monate dauern kann. Die erwünschte Langzeitfreisetzung von Lipoplexen, die in die PLGA-Beschichtungen eingebettet waren, konnte vom ersten bis zum zwanzigsten Tag gezeigt werden. Die etablierte Atelokollagen-Beschichtung mit siICAM–1-Lipoplexen bewirkte eine signifikante Reduktion der ICAM–1-Expression bis zum achten Tag. In beiden Ansätzen wurde die spezifische Spaltung der ICAM–1 messenger RNA (mRNA) gezeigt und eine gute Zellviabilität und Hämokompatibilität der Beschichtungen nachgewiesen. Der PLGA-Mehrschichtaufbau eignet sich außerdem zur simultanen Kotransfektion der grün fluoreszierenden (eGFP)mRNA und siRNA. Die beiden substratvermittelten siRNA-Freisetzungssysteme mit Langzeitwirkung ebnen den Weg für neue therapeutische Stentbeschichtungen, um das ISR-Risiko nach der Stentimplantation zu reduzieren. Das PEM-System mit HA und Polyplexen zeigte eine schnelle Freisetzung von siRNA-Partikeln innerhalb der ersten Stunde. Eine signifikante Verringerung des ICAM–1 Proteins wurde nachgewiesen und die Zellviabilität war nicht signifikant reduziert. Somit liefert diese Entwicklung ein weiteres lokales Anwendungssystem, das in die PTCA integriert werden kann

Development of Therapeutic-Grade Small Interfering RNAs by Chemical Engineering

Recent successes in clinical trials have provided important proof of concept that small interfering RNAs indeed constitute a new promising class of therapeutics. Although great efforts are still needed to ensure efficient means of delivery in vivo, the siRNA molecule itself has been successfully engineered by chemical modification to meet initial challenges regarding specificity, stability and immunogenicity. To date, a great wealth of siRNA architectures and types of chemical modification are available for promoting safe siRNA mediated gene silencing in vivo and, consequently, the choice of design and modification types can be challenging to individual experimenters. Here we review the literature and devise how to improve siRNA performance by structural design and specific chemical modification to ensure potent and specific gene silencing without unwarranted side effects and hereby complement the ongoing efforts to improve cell targeting and delivery by other carrier molecules

Gene silencing of cystatin B (CSTB) by RNAi : Implications for the altered JAK/STAT signaling pathway in Unverricht-Lundborg disease (EPM1)

Unverricht-Lundborg disease (EPM1/ULD, OMIM 254800) is an autosomal recessive inherited severe type of epilepsy with myoclonus and progressive neurological degeneration. The incidence of EPM1 in Finland is 1:20.000 births per year, and there are about 200 diagnosed cases. The age of disease onset is between 6 and 16 years. The symptoms start with epileptic seizures, stimulus sensitive myoclonus, and generalized tonic-clonic seizures, and progress within a few years to ataxia, incoordination, and dysarthria. Fourteen EPM1-associated loss-of-function mutations in the gene cystatin B (CSTB) have been described. CSTB is a ubiquitously expressed intracellular cysteine proteinase inhibitor, counteracting i.e. cathepsins B, L, and K. The expression levels of CSTB are higher in cerebellar Purkinje cells and in Bergmann glia of the adult central nervous system. There is a Cstb-deficient mouse model for EPM1, which shows progressive death of neurons and widespread gliosis. It has been earlier shown that Cstb knockdown sensitizes cerebellar granule neurons to cathepsin B mediated oxidative stress, resulting in cell death. This master's thesis is based on a previously done gene expression profiling of primary microglia of Cstb-/- mice, which revealed a downregulation of type I and type II interferon-regulated genes on the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) –signaling pathway. The two aims of this study were to create an in vitro disease model for EPM1 in the human cervical adeno-carcinoma cell line HeLa, and in the murine leukemic cell line RAW264.7 by siRNA mediated RNA inhibition, and to study the effects of Cstb knockdown in selected interferon regulated genes of the JAK/STAT signaling pathway. Cystatin B was successfully knocked down in both cell lines HeLa and RAW264.7, and the obtained kinetics of Cstb knockdown in the cell line RAW264.7 provided with valuable information for the sec-ond part of the study. In the cell line HeLa, downregulation of CSTB did not change the expression lev-els of the genes of the JAK/STAT signaling pathway. In the cell line RAW264.7, CSTB knockdown on protein level was followed by a downregulation of the genes Stat1, Stat2, and Irf9. These results were in concordance with the results that had been obtained from the previously performed gene expression profiling of Cstb-/- microglia

RNAi-mediated Reduction of a Major Ruminant Specific Milk Allergen Using a Transgenic Mouse Model

Milk from dairy cows is an important human food. It contains the protein beta-lactoglobulin (BLG) which is not present in human milk and thought to be a major allergen in the milk of cows. RNA interference (RNAi), mediated by sequence-specific micro RNAs (miRNAs), provides a new molecular tool to reduce the levels of undesirable proteins. In this study, I have screened ten miRNAs targeting the BLG mRNA for their potential to knockdown BLG expression using a cell-based in vitro assay. I identified four miRNAs which showed substantial bovine BLG (bBLG) (78-97%) and ovine BLG (oBLG) (33-94%) knockdown activity in vitro. Tandem miRNA constructs with combinations of these four single miRNAs did not result in significantly increased knockdown efficiency compared to the respective single miRNA constructs in the in vitro assay (P>0.05). Because targeting of two different regions has the potential for greater knockdown efficiency in vivo, I chose a tandem construct which achieved in vitro up to 99% bBLG knockdown as compared to 92% and 74% of bBLG knockdown by the single miRNAs. The tandem construct also showed 90% of oBLG knockdown in comparison to 92% and 69% of oBLG knockdown by the single miRNAs in the in vitro assay which made it an ideal candidate for the subsequent evaluation in transgenic (Tg) mice. For the in vivo studies, the cytomegalovirus (CMV) promoter of the selected miRNA constructs was replaced with the promoter of the mouse milk protein gene for whey acid protein (WAP) which directs the expression of the BLG-specific miRNAs to the lactating mammary gland. The Tg mouse lines generated with the tissue-specific tandem construct and the single miRNAs used for the tandem miRNA construct were then crossed with an oBLG or bBLG expressing Tg mouse line to generate double transgenic (miRNA-BLG) mice for assessing BLG knockdown in vivo. Analysis of the milk proteins demonstrated that the tandem miRNA reduced the levels of oBLG and bBLG in milk from the miRNA-BLG mice up to 96% and 46%, respectively. This study validates the mammary gland specific expression of miRNAs as a promising approach to knockdown allergenic proteins in milk

Chemical genetic dissection of efferent IRE1α signalling

The Endoplasmic Reticulum is the cellular organelle primarily responsible for producing proteins on the secretory pathway, a pathway important in the production of biopharmaceuticals. One of the requirements for the successful production of a functional protein is correct folding of the polypeptide sequence. During conditions such as viral infection, mutant protein expression and cell differentiation the endoplasmic reticulum is placed under conditions of stress. IRE1 is a protein kinase and endoribonuclease, which along with PERK and ATF6, forms part of the Unfolded Protein Response, the system by which the cell deals with the stress caused by a high protein load. IRE1 is capable of increasing the protein folding capacity of the ER, by upregulating chaperone proteins and reducing the load by attenuating translation, (protective response). This action is mediated by splicing of the mRNA coding for the bZIP transcription factor XBP-1. IRE1 is also capable of causing apoptotic responses via TRAF2 (cell injuring response) resulting in the activation of JNK and NFκB. In this study, using site directed mutagenesis a panel of IRE1 mutants was produced and screened for alterations to the protective and cell injuring responses. Of these the D711A mutant was shown in mouse embryonic fibroblasts to retain endoribonuclease activity, and to display an attenuated cell injuring response. When this mutant was applied to an industrial CHO cell line it appeared to exhibit an increase in biopharmaceutical productivity over the wild type IRE1, indicating its potential for use in the biopharmaceutical cell lines

Translational control of Human Epidermal Growth Factor Receptors, HER2 and HER4 in response to cellular stresses in breast cancer cells

The human epidermal growth factors (EGFR and HER2-4) are tyrosine kinase cell surface receptors that play key roles in cell signalling, growth, and differentiation. However, HER2 and HER4 are commonly overexpressed in breast cancer contributing to breast cancer aggressiveness. Translational mechanisms such as internal ribosome entry sites (IRESs) and upstream opening reading frames (uORFs) can mediate translation of specific mRNA under cell stress conditions when global translation is inhibited. Cancer cells can be exposed to many cell stress conditions such as hypoxia and nutrient starvation, and such translational control mechanisms can contribute to overexpression of mRNAs. EGFR mRNA contains an IRES in the 5’ untranslated region (5’ UTR) that maintains expression of a firefly luciferase reporter in response to stress conditions such as hypoxia. However, it is not known if the 5’ UTRs of the other HER family members (HER 2-4) contain an IRES. Additionally, HER2 mRNA contains a repressive uORF in the 5’ UTR and a translational derepression element (TDE) in the 3’ UTR. However, it has not been investigated whether HER2 5’ UTR can mediate an increase in translation in response to glutamine or glucose starvation as seen for ATF4 and GCN4 mRNA. To screen for IRES activity in HER2 or HER4 5’ UTRs, bicistronic luciferase constructs were created that contained the 5’ UTR of HER2 or HER4 between two luciferase reporters. In contrast to EGFR 5’ UTR, there was no evidence of IRES activity in the 5’ UTRs of HER2 and HER4 in response to endoplasmic reticulum stress, oxidative stress, low serum conditions, a hypoxia mimic, genotoxic stress, confluence stress, glutamine starvation and glucose starvation in MCF-7 breast cancer cells. Additionally, monocistronic constructs were created that contained the 5’ UTRs of HER2 or HER4 upstream to a single firefly luciferase reporter. In monocistronic constructs, HER2 and HER4 5’ UTRs did not increase translation in response to glutamine or glucose starvation in MCF-7 cells. Finally, in contrast to a previous publication, the TDE in HER2 3’ UTR did not derepress translation under non-stressed conditions or additionally in response to glutamine or glucose starvation. The work in this thesis investigated the translational control of important oncogenes in stress conditions that are biologically relevant to cancer. Despite not identifying any translational control mechanisms in HER2 or HER4 5’ UTRs, the work in this thesis provides a promising approach for characterising the translational control of other important oncogenes that could provide potential therapeutic targets for cancer therapy in the future