Sirna And Mirna Gene Silencing From Bench To Bedside Pdf
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- Current Development of siRNA Bioconjugates: From Research to the Clinic
- siRNA and miRNA Gene Silencing
- siRNA and miRNA Gene Silencing
- MicroRNAs and Cancer: From Bench to Bedside
Current Development of siRNA Bioconjugates: From Research to the Clinic
Through protein complex purification and spectrometry MS analyses many factors involved in this process have been identified and reported. These mutations were shown to affect microRNA processing, dicer function and promote tumorigenesis. Consistent with the notion that an overall down-regulation of miR levels in cancer contributes significantly to the progression of the disease we showed that while a decrease in Dicer expression increase the transforming potential of cancer cell lines complete silencing is not tolerated by most cancer cells.
These data were further supported by genetic experiments in mice. We indeed showed that Dicer functions as a haploinsufficient tumor suppressor gene in a pre-clinical mouse model of retinoblastoma but that it is in the same time required for tumorigenesis. Together these observations have opened exciting new avenues for anticancer therapy. Another important objective is the identification of novel putative cancer-causing ncRNAs. On a technological point of view, in order to further facilitate and improve sensitivity and accuracy of genome-wide ncRNA profiling, we generated new microarray platforms based on the LNA technology.
In addition, promiscuous LNA oligos have been designed in attempts to facilitate the detection or inhibition of entire miRNA families rather than single members of a given family as well as long non-coding RNAs. These so-called target site blockers have given very promising results and are now commercially available at Exiqon. In order to define the role of selected ONCOMIRS, lncRNAs and components of the miR-processing machinery in the genesis and progression of cancer, classical gain and loss of function approaches in vivo, using the mouse as a model system were performed.
We for instance showed that complete loss of miR completely prevents retinoblastoma formation in mice Nittner et al. In contrast loss of miR did not affect normal retinogenesis; an observation that has important therapeutic implications. In contrast, however, although upregulated in mouse and human melanoma the miR cluster was shown to be dispensable for NRas-induced melanoma formation in mice.
A new generation of miR mimics and antisense therapeutics have developed. We have tested and optimised the biostability, cytotoxicity and electropulsation-based delivery in vivo of such molecules and demonstrated that electrotransfer of miR inhibitors offers a new avenue for anti-cancer retinoblastoma and glioblastoma therapy.
Period 1 Recent experiments have indicated that alterations in miR levels in cancer could be partly the result of aberrant post-transcriptional processing of precursor miR transcripts. In the last few years, partner 2 has isolated two complexes required for the sequential processing of primary miR transcript into the mature miR in human cells.
We proposed to actively persue the identification of additional components involved in the regulation of silencing by miRs, through protein complexes purification. We proposed to implement the LNA technology present in the group partner 6 on high-density flexible array platforms to facilitate ncRNA expression analyses.
Once this technology will be available, a more long-term goal will be the identification of novel small non-coding RNAs ncRNAs, including miRs whose expression is dysregulated in human cancers. In the meantime, we proposed to establish ncRNA gene expression profiles using currently available technologies with the aim to identify genes that are regulated by and therefore act as downstream mediators of the p53 tumor suppressor pathway.
Only a limited number of oncomiR-target pairs have been identified to date. Partner 4 has recently developed a new approach for miR targets identification. We proposed to use this approach in combination with genome-wide gene expression profiling in order to identify the primary and secondary targets of a number of selected ONCOMIRS.
To this end, we proposed to generate classical gene targeting vectors that disrupt expression of the miR s of interest in mice. For conditional gain of function studies, we will also knock-in miR gene s into the ROSA26 locus by gene targeting. The newly generated alleles will be combined with tumour-causing mutations to study the consequence of aberrant expression of a particular miR in relevant tumor models of human cancers.
RNA interference represents a potential strategy for in vivo target validation, therapeutic product development and clinical new technologies. It is expected to be particularly useful to silence cancer-causing genes that encode targets that are not amenable to conventional therapeutics. Moreover, there is evidence that miR-based molecules enter the RNAi pathway through a more natural route and yield more effective silencing with reduced toxicity and off-target effects.
One of our main objectives is to design miR-based molecules miR mimics that either allow potent activation of a 'dormant' tumour suppressor pathway for instance p53 or alter expression of specific oncogenes. The molecules will be designed to allow efficient delivery, high biostability and low toxicity. The LNA technology developed by partner 6 will be particularly useful in this context. We first planned to test the efficacy and toxicity of these compounds in culture cells.
Period 2 Recent experiments have indicated that alterations in miR levels in cancer could be partly the result of aberrant post-transcriptional processing of precursor miR transcripts. We proposed to actively pursue the identification of additional components involved in the regulation of silencing by miRs, through protein complexes purification.
Human tumours are characterised by widespread reduction in microRNA miRNA expression, although it is unclear how such changes come about and whether they have an etiological role in the disease. A defect in miRNA-processing is one possible mechanism for the global down-regulation.
To explore this possibility in more detail in vivo we proposed to manipulate Dicer1 gene dosage in a mouse model of retinoblastoma and identify miRs that can either function as tumour suppressors or oncogenes in such a model. A more long-term goal was the identification of novel small non-coding RNAs ncRNAs, including miRs whose expression is dysregulated in human cancers.
In the meantime, we proposed to establish ncRNA gene expression profiles using currently available technologies with the aim to identify genes that are regulated by and therefore act as downstream mediators of the p53 tumour suppressor pathway. The newly generated alleles will be combined with tumour-causing mutations to study the consequence of aberrant expression of a particular miR in relevant tumour models of human cancers.
Period 3 Recent experiments have indicated that alterations in miR levels in cancer could be partly the result of aberrant post-transcriptional processing of precursor miR transcripts. Partner 4 and 7 have recently developed new approaches for miR targets identification.
To this end, we proposed to generate miRKO and transgenic mouse lines and combined these alleles with tumour-causing mutations to study the consequence of aberrant expression of selected miRs in relevant tumor models of human cancers. Period 4 last Recent experiments have indicated that alterations in miR levels in cancer could be partly the result of aberrant post-transcriptional processing of precursor miR transcripts.
We proposed to actively search for additional components involved in the regulation of silencing by miRs, through protein complexes purification. Human tumors are characterised by widespread reduction in microRNA miRNA expression, although it is unclear how such changes come about and whether they have an etiological role in the disease.
To explore this possibility in more detail in vivo we proposed to manipulate Dicer1 gene dosage in a mouse model of retinoblastoma and identify miRs that can either function as tumor suppressors or oncogenes in such a model.
We proposed to implement the LNA technology present in the group partner 6 as a tool to knock-down expression of selected non-coding RNAs. To this end, we generated KO and transgenic mouse lines for miRs and lncRNAs and combined these alleles with tumour-causing mutations to study the consequence of aberrant expression of selected miRs in relevant tumour models of human cancers.
One of our main objectives was to design miR-based molecules miR mimics that either allow potent activation of a 'dormant' tumour suppressor pathway for instance p53 or alter expression of specific oncogenes. Such molecules have be designed to allow efficient delivery, high biostability and low toxicity. The LNA technology developed by partner 6 has been particularly useful in this context.
Drosha processing yields miRNA precursors pre-miRNAs , which are subsequently transported to the cytoplasm by the export receptor exportin 5. In human somatic cells, Ago1, Ago2, Ago3 and Ago4 are expressed. It is likely that all four Ago proteins are incorporated into similar protein complexes. One of our objectives was the identification of novel components of the miR-processing machinery, through protein complexes purification.
Partner 2 identified the huntingtin Htt protein as a component of the Argonaute complex. Colocalisation studies demonstrated Htt and Ago2 to be present in P bodies, and depletion of Htt showed compromised RNA-mediated gene silencing.
Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an extension of polyglutamines in the Htt protein. The data therefore suggest that this disease may be attributed in part to mutant Htt's role in post-transcriptional processes Savas et al. In close collaboration with Elisabeth Kremmer Helmholtz center, Munich , beneficiary 7 generated monoclonal antibodies against a number of proteins, involved in miRNA function.
Antibodies against individual human Ago proteins have thus been generated. One of these antibodies allows detection of endogenous Ago2 expression on tissue sections. Beneficiary 7 isolated a number of interesting Drosha protein partners among them EWS. Partner 7 also established a list of Dicer-dependent and -independent AGO2-binding proteins Frohn et al. Beneficiary 7 has confirmed the presence of the helicase p68 in the DROSHA complex; an observation which is consistent with previously published studies.
Strikingly, genetic data in C. A set of proteins associates with Ago2 in an mRNA- independent manner. Taken together, this mass spectrometry approach revealed that Ago2 associates with larger RNA species even in the absence of small RNAs. To functionally validate the interaction data, Partner 7 used luciferase-based miRNA reporters. In addition, we employed a reporter containing the Hmga2 3'-UTR with mutated let-7a target sites and normalised the data against each other.
As expected, Ago2 knock down led to increased luciferase activity. Knock down of EDC4, which has been implicated in miRNA function in Drosophila, resulted in specific luciferase up-regulation as well, suggesting that EDC4 is indeed involved in silencing of the Hmga2 reporter construct.
Since ZNF is a putative transcription factor, it is possible that it cooperates with Ago2 in nuclear Ago functions. The previously recognised role of let-7 family members as tumour suppressors prompted us to assess the role of PRS14 and EDC4 in cellular transformation by performing colony and soft agar assays.
Moreover we have also assessed the role of FXR2 in cellular transformation, as other Fragile X mental retardation proteins have recently been implicated in cancer development. These experiments have confirmed that all these factors are able to modulate cellular transformation in these assays.
Further experiments aimed at dissecting the mechanisms underlying these phenotypic consequences are ongoing in the laboratory of partner 1 and 7. Overall, down-regulation of miR levels in cancer contributes significantly to the progression of the disease. Two heterozygous frame-shift mutations were identified in two different cell lines.
These mutations were found to cause a significant decrease in TRBP expression. Importantly, ectopic expression of wild-type TRBP, but not of the naturally occurring mutants, induced tumour suppressor-like features in colorectal cancer cell lines.
Notably, Dicer overexpression also showed tumour suppressor properties as determined using classical tissue culture assays MTT and colony assays and mouse xenograph models nude mice. The above findings provide further evidence of a role of loss of function events in the regulation of miRNA processing machinery during tumourigenesis.
However, the molecular mechanisms that promote tumourigenesis as a consequence of global repression of miRNA maturation remain elusive. Data obtained by beneficiary 1 suggest that the p53 tumor suppressor pathway is compromised in cancer cells harbouring hypomorphic Dicer expression. Given the results of the mutational analyses of the various components of the miR-processing machinery in cancer cell lines and primary tumours, it was decided to slightly deviate from the initial plans.
To add relevance to our structure function analysis, the experiments were conducted in colorectal cancer cell lines, instead of NIH-3T3, since these mutations were found to occur in this particular tumour context. Moreover, since no mutations were found in Dicer and AGO2 in human tumours, construction of the initially proposed Dicer and AGO2 mutants has been cancelled.
Instead, we have already tested the role of TRBP and the influence of the naturally occurring mutations to cell transformation.
siRNA and miRNA Gene Silencing
These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Alnylam Pharmaceuticals. No additional funding was received for this study. Alnylam employees were collaborators on this project. Alnylam Pharmaceuticals had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Skip to search form Skip to main content You are currently offline. Some features of the site may not work correctly. DOI: Leung and W. Liang Published Biology, Medicine Molecules.
PDF | On Jun 1, , Francesco Nicassio published Mouldy Sioud (ed): siRNA and miRNA Gene Silencing: from Bench to Bedside | Find.
siRNA and miRNA Gene Silencing
Clin Sci Lond 13 November ; 21 : — We have known for just over a decade that functional RNA is shuttled between cells Nat. Cell Biol.
MicroRNAs and Cancer: From Bench to Bedside
The main problems in the development of siRNA-based drugs for therapeutic use are the low efficiency of siRNA delivery to target cells and the degradation of siRNAs by nucleases in biological fluids. Various approaches have been proposed to solve the problem of siRNA delivery in vivo e. One of the most promising approaches to solve the problem of siRNA delivery to target cells is bioconjugation; i. This review is focused on strategies and principles for constructing siRNA bioconjugates for in vivo use. Chemical modifications of RNA have been developed that modulate their activity and stabilize them in biological fluids Hoerter and Walter, ; some progress has been made in the development of methods for the delivery of siRNAs to cells Hassler et al. Several siRNA-based drugs are undergoing clinical trials, and one drug patisiran Onpattro is approved for use in the clinic Garber, However, the outstanding potential of siRNAs as therapeutic drugs has not yet been fully implemented.
Download full-text PDF General mechanism of action for siRNA induced gene silencing. molecules from bench to bedside. There are several types of RNAi therapeutics: micro RNA (miRNA), short hairpin (shRNA).
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На противоположной стене висело распятие в натуральную величину. Беккер остановился. Тупик.