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Development of whole body scanning PCR, a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides

Boos, Julien Alfred Jean-Paul. Development of whole body scanning PCR, a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides. 2015, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_11433

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Abstract

Based on RNA interference (RNAi), a mechanism regulating gene expression at the post-transcriptional level in eukaryotes, therapeutic siRNAs are slowly moving towards late phase clinical trials. The development of therapeutic oligonucleotides is however paved with many challenges, mostly regarding a mean to deliver siRNAs in a highly tissue-specific manner and to monitor their in vivo biodistribution. Two major axes are therefore structuring this thesis, the first one being the development of a method to assess the in vivo biodistribution of siRNAs, and the second one aiming at unraveling the possible use of exosomes for tissue-specific delivery.
We first developed an assay compatible with various RT-qPCR assays to assess the biodistribution of oligonucleotides biodistribution in mouse whole body sections. The RT-qPCR signals are converted into an image that represents the in vivo biodistribution of the analyzed oligonucleotide. This method, called Whole Body Scanning PCR (WBS-PCR), has been first validated for the detection of tissue-specific or ectopic mRNAs. We subsequently developed an assay to detect miRNAs and siRNAs by RT-qPCR, which was validated with the WBS-PCR for the detection of tissue-specific miRNAs. An in vivo study was then performed, aiming at determining the in vivo biodistribution of an unformulated siRNA in mouse whole body sections. However, since more and more chemistry is being incorporated in therapeutic oligonucleotides to provide them higher resistance towards nucleases, these oligonucleotides could not be detected anymore by our assay. We therefore designed a novel assay, allowing the detection of highly chemically modified oligonucleotides, which we called Chemical Ligation qPCR (CL-qPCR). The compatibility of CL-qPCR with WBS-PCR was assessed by performing in vivo studies using a fully 2’-MOE modified antagomir, an oligonucleotide targeting miR-16-5p.
Having successfully developed a method allowing the rapid determination of the in vivo biodistribution of therapeutic oligonucleotides, we focused in the second part of this thesis on assessing the use of exosomes as novel oligonucleotides delivery vehicles.
Although a vast majority of siRNAs in clinical trials are delivered systemically via lipidic nanoparticles (LNP), some concerns have been raised with respect to the difficulty to efficiently design and generate LNPs displaying tissue-specificity and low toxicity. An increasing amount of studies reported that naturally occurring vesicles called exosomes might be involved in inter-cellular communication via the transfer of proteins, mRNAs and miRNAs, suggesting some degree of tissue-specific delivery potential. Moreover, the fact that these exosomes are secreted by cells, and their presence in the circulation indicates that these vesicles might exhibit a low lipid-induced toxicity, made them ideal transporters candidates of therapeutic oligonucleotides. We first investigated ways to efficiently characterize exosomes isolated from cell culture supernatant. An extensive characterization of exosomes was crucial because of the subsequent in vivo injections, planned to demonstrate the utility of exosomes as oligonucleotides delivery vehicles. We therefore selected a series of criteria which for us accounted for the presence of exosomes in a sample. These criteria are based on exosomes hallmarks such as particle size, the detection of exosomes-enriched miRNAs, as well as the detection of exosomal proteins. We then focused on the identification of an efficient exosomes isolation method from cell culture supernatant. We therefore compared various methods described in the literature for exosomes isolation, such as the commonly used differential ultracentrifugation, or the recently commercialized ExoQuick kit. We also tested isolation methods based on either particle size segregation, or taking advantage of the virus-like characteristics of exosomes.None of the samples prepared by using these methods did meet all criterions which could have confirmed the presence of exosomes in the purified cell culture supernatants. However, further characterization of one of the exosomes isolation methods confirmed that cells might not only secrete miRNAs encapsulated in exosomes, but also miRNAs in complex with a member of the RISC complex, namely Ago2.
Although CL-qPCR, in combination with WBS-PCR, would allow the detection and biodistribution of many kinds of unmodified, or heavily chemically modified therapeutic siRNAs, we were unable to demonstrate the use of exosomes as siRNA delivery vehicles.
Advisors:Hynes, Nancy
Committee Members:Beuvink, Iwan and Grosshans, Helge
Faculties and Departments:09 Associated Institutions > Friedrich Miescher Institut FMI
Item Type:Thesis
Thesis no:11433
Bibsysno:Link to catalogue
Number of Pages:104 S.
Language:English
Identification Number:
Last Modified:31 Aug 2017 10:16
Deposited On:04 Dec 2015 11:06

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