Characterization of Sirt2 using conditional RNAi in mice

Within the past eight years, RNA interference (RNAi) has emerged as a powerful experimental tool for gene function analysis in mice. Reversible control of shRNA mediated RNAi has been achieved by using a tetracycline (tet)-inducible promoter. In the presence of the inductor doxycycline (dox), shRNA mediated gene silencing is initiated, whereas RNAi mechanism is blocked in the absence of dox. To achieve spatially and temporally regulated RNAi, the tet inducible system was combined with a Cre/loxP based strategy for tissue specific activation of shRNA constructs. To this end, a loxP-flanked "promoter inhibitory element" (PIE) was placed between the proximal (PSE) and distal sequence element (DSE) of a dox inducible promoter such that promoter function is completely blocked. Re-activation can be achieved through Cre mediated excision of PIE. To allow for gene silencing in a selected tissue, Cre expression can be regulated by a tissue-specific promoter. In mouse ES cells, the system mediated tight regulation of shRNA expression upon Cre mediated activation and dox administration, reaching knockdown efficiencies of >80%. Unexpectedly, the system showed a limited activity in transgenic mice when applied for conditional silencing of two different targets, LacZ and Sirt2. Sirt2 is a member of the sirtuin family which has considerably gained attention in vitro for its possible role in many physiological processes, including adipogenesis and neurodegenerative diseases. To investigate the function of Sirt2 in vivo, the unmodified dox-responsive and tet-inducible promoter was further used for conditional RNAi in transgenic mice. Inducible shRNA expression resulted in efficient silencing of Sirt2 (>90%) in all tissues which have been analyzed. Suppression of Sirt2 during embryogenesis resulted in offspring consisting of equal ratios of wild type and transgenic pups, indicating that Sirt2 is not indispensable for development. In adult animals, glucose metabolism, insulin sensitivity and energy balance appeared to be unaffected by Sirt2 deficiency. Likewise, expression of PPARγ, a downstream target of Sirt2, was not found to be altered upon Sirt2 inhibition. Finally, Sirt2 silencing was induced in an experimental model of Parkinson disease (PD). Data from Rotarod performances to study motor behaviour did not provide any evidence for a role of Sirt2 in PD pathogenesis as suggested by previous in vitro studies. Taken together, conditional Sirt2 silencing in vivo does not support speculation concerning a central role of Sirt2 in physiological processes, embryogenesis and in a mouse model of Parkinson disease

Lentiviral manipulation of gene expression in human adult and embryonic stem cells

Human stem cells could revolutionize the field of medicine by providing a diverse range of cell types for tissue replacement therapies and drug discovery. To achieve this goal, genetic tools need to be optimized and developed for controlling and manipulating stem cells ex vivo. Here we describe a lentiviral delivery system capable of high infection rates in human mesenchymal and embryonic stem cells. The lentiviral backbone was modified to express mono- and bi-cistronic transgenes and was also used to deliver short hairpin ribonucleic acid for specific silencing of gene expression in human stem cells. We show that lentiviral transduction can be used to alter gene expression without altering the genes' ability to differentiate in vitro. These vectors will enable rapid analysis of gene function in stem cells and permit the generation of knock-in / knock-out models of human disease in the rapidly developing field of gene therapy

Development of replication-defective herpes simplex viral vectors for delivery of RNA interference to neurons of the peripheral nervous system

Considerable interest has been focused on inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although small interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi to the central and peripheral nervous system remains a major challenge limiting its applications. This thesis describes the development of a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) using replication-defective herpes simplex viral (HSV-1) vectors by identifying and evaluating various approaches to induce RNAi, i.e. expression of individual short-hairpin RNAs (shRNAs), artificial microRNAs (miRNAs) and multiple tandem miRNAs. Following the development of these systems, HSV-mediated delivery of shRNA or miRNA against reporter genes was shown to result in highly effective and specific silencing in neuronal and non-neuronal cells in culture and in the DRG of mice in vivo, including in a transgenic mouse model. Proof of concept was established by demonstrating in vivo silencing of the endogenous trpv1 gene, thought to be involved in nociception, by assessing both mRNA and protein levels. These data are the first to show silencing in DRG neurons in vivo by vector-mediated delivery of shRNA and support the utility of HSV vectors for gene silencing in peripheral neurons and the potential application of this technology to the study of nociceptive processes and in pain gene target validation studies. Moreover, a disabled HSV-1 vector targeting p75, Lingo1 and NgR2, which are involved in myelin inhibition of axonal regeneration, was developed and evaluated for its ability to promote regeneration of sensory axons into the spinal cord, following injury of the dorsal roots. This is the first time such an appoach to silencing multiple genes has been employed. Although HSV-mediated delivery of multiple miRNAs resulted in highly effective silencing of these genes in dividing cells in culture, while highly effective silencing of p75 was achieved, only modest silencing of Lingo1 and NgR2 was observed in DRG neurons in vivo. Preliminary regeneration experiments, which were largely outside the scope of this thesis, were inconclusive and require more extensive study as a stand-alone project, if the in vivo potential of the approach developed for silencing multiple genes targeted at axonal regeneration is to be further explored

A Re-Examination of Global Suppression of RNA Interference by HIV-1

The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing

Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines

Conditional expression of short hairpin RNAs (shRNAs) to knock down target genes is a powerful tool to study gene function. The most common inducible expression systems are based on tetracycline-regulated RNA polymerase III promoters. During the last years, several tetracycline-inducible U6 and H1 promoter variants have been reported in different experimental settings showing variable efficiencies. In this study, we compare the most common variants of these promoters in several mammalian cell lines. For all cell lines tested, we find that several inducible U6 and H1 promoters containing single tetracycline operator (tetO) sequences show high-transcriptional background in the non-induced state. Promoter variants containing two tetO sequences show tight suppression of transcription in the non-induced state, and high tet responsiveness and high gene knockdown efficiency upon induction in all cell lines tested. We report a variant of the H1 promoter containing two O2-type tetO sequences flanking the TATA box that shows little transcriptional background in the non-induced state and up to 90% target knockdown when the inducer molecule (dox–doxycycline) is added. This inducible system for RNAi-based gene silencing is a good candidate for use both in basic research on gene function and for potential therapeutic applications

RNAi for Treating Hepatitis B Viral Infection

Chronic hepatitis B virus (HBV) infection is one of the leading causes of liver cirrhosis and hepatocellular carcinoma (HCC). Current treatment strategies of HBV infection including the use of interferon (IFN)-α and nucleotide analogues such as lamivudine and adefovir have met with only partial success. Therefore, it is necessary to develop more effective antiviral therapies that can clear HBV infection with fewer side effects. RNA interference (RNAi), by which a small interfering RNA (siRNA) induces the gene silence at a post-transcriptional level, has the potential of treating HBV infection. The successful use of chemically synthesized siRNA, endogenous expression of small hairpin RNA (shRNA) or microRNA (miRNA) to silence the target gene make this technology towards a potentially rational therapeutics for HBV infection. However, several challenges including poor siRNA stability, inefficient cellular uptake, widespread biodistribution and non-specific effects need to be overcome. In this review, we discuss several strategies for improving the anti-HBV therapeutic efficacy of siRNAs, while avoiding their off-target effects and immunostimulation. There is an in-depth discussion on the (1) mechanisms of RNAi, (2) methods for siRNA/shRNA production, (3) barriers to RNAi-based therapies, and (4) delivery strategies of siRNA for treating HBV infection

Lentiviral vectors encoding tetracycline-dependent repressors and transactivators for reversible knockdown of gene expression: a comparative study

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi)-mediated by the expression of short hairpin RNAs (shRNAs) has emerged as a powerful experimental tool for reverse genetic studies in mammalian cells. A number of recent reports have described approaches allowing regulated production of shRNAs based on modified RNA polymerase II (Pol II) or RNA polymerase III (Pol III) promoters, controlled by drug-responsive transactivators or repressors such as tetracycline (Tet)-dependent transactivators and repressors. However, the usefulness of these approaches is often times limited, caused by inefficient delivery and/or expression of shRNA-encoding sequences in target cells and/or poor design of shRNAs sequences. With a view toward optimizing Tet-regulated shRNA expression in mammalian cells, we compared the capacity of a variety of hybrid Pol III promoters to express short shRNAs in target cells following lentivirus-mediated delivery of shRNA-encoding cassettes.</p> <p>Results</p> <p>RNAi-mediated knockdown of gene expression in target cells, controlled by a modified Tet-repressor (TetR) in the presence of doxycycline (Dox) was robust. Expression of shRNAs from engineered human U6 (hU6) promoters containing a single tetracycline operator (TO) sequence between the proximal sequence element (PSE) and the TATA box, or an improved second-generation Tet-responsive promoter element (TRE) placed upstream of the promoter was tight and reversible as judged using quantitative protein measurements. We also established and tested a novel hU6 promoter system in which the distal sequence element (DSE) of the hU6 promoter was replaced with a second-generation TRE. In this system, positive regulation of shRNA production is mediated by novel Tet-dependent transactivators bearing transactivation domains derived from the human Sp1 transcription factor.</p> <p>Conclusion</p> <p>Our modified lentiviral vector system resulted in tight and reversible knockdown of target gene expression in unsorted cell populations. Tightly regulated target gene knockdown was observed with vectors containing either a single TO sequence or a second-generation TRE using carefully controlled transduction conditions. We expect these vectors to ultimately find applications for tight and reversible RNAi in mammalian cells in vivo.</p

Targeted MYCN suppression and its effect on miRNA in neuroblastoma

Neuroblastoma is the most common extra-cranial tumour disease in children, and accounts for 15% of all childhood cancer deaths. MYCN is a transcription factor and a proto-oncogene that most often initiates transcription of target genes involved in increased proliferation and inhibition of differentiation. MYCN amplified neuroblastoma represents the most aggressive form of this disease. Silencing of MYCN is an attractive approach for understanding the role of this gene in development and disease. MYCN is involved in development, proliferation and differentiation. In order to explore its functions in detail, it is a great advantage to be able to silence its expressions in all cells present for an indefinite period of time, and at any suitable time-point. In paper I we describe a novel design of an inducible H1 promoter capable of delivering shRNA in a conditional manner. This versatile system allows inducible expression of any desired shRNA. In paper II this promoter is used to control the conditional expression of anti-MYCN shRNAs from cassettes stably integrated in two MYCN-amplified cell lines. Here MYCN is silenced when the inducer doxycycline is added to the media, resulting in increased differentiation and reduced proliferation of the MYCN amplified cell lines. MiRNAs are small non-coding RNA shown to regulate the expression of a vast amount of proteins in humans. In paper III we investigate the effect of MYCN suppression on the global miRNA expression in MYCN-amplified neuroblastoma. We find that reduced MYCN expression leads to both increased and reduced expression of several miRNAs. The function of mir-21 is investigated further, and no effect on neither proliferation nor differentiation is found. Other miRNAs demonstrated to be regulated by MYCN, is mir-92a and mir-92b. In paper IV we demonstrate how these miRNAs suppresses the expression of DKK3, a tumour suppressor gene often found to be down-regulated in MYCN-amplified neuroblastoma

A Novel Approach of Virotherapy Based Hsf-1 Shrna in Cancer Eradication

Cancer is the second leading cause of death worldwide with continues rise mortality rate. Current cancer treatment modalities are still ineffective and associated with many side effects leading to robust research to find new specific target therapy. Heat shock factor (HSF)-1 is heat shock response mediator protein and act as transcription factor for HSP encoding gene. Many cancers have up-regulated HSP as a result of increase HSF-1 expression. Interestingly, inhibition of HSF-1 has no effect to normal cell, indicating HSF-1 as promises target therapy. RNAi is potential mechanism to block and down regulate HSF-1 which will affect many cellular processes in cancer cell. Combining RNAi base treatment with oncolityc viruses will boost the therapeutic effect of this novel treatment. Despite its potency, this modality still need further research in order to evaluate its efficacy and optimal doses to gain optimal result