Non-coding RNAs (e.g. micro-RNAs) function as relevant signaling molecules in controlling cell fate. Since these molecules are loaded into extracellular vesicles (exosomes), they also deeply impact cell-to-cell communication.
This project builds on our previous results published in Santangelo et al., Cell Reports 2016: in this work we demonstrated that a conserved extra seed sequence shared by a subset of miRNAs enriched in hepatocyte-derived exosomes has a crucial role in the regulation of their exosome sorting (by means of direct interaction with the RNA-binding protein SYNCRIP).
We deem that it is possible to convey the conceptual advantage resulting from these data into innovative strategies establishing ncRNAs as tools for targeting relevant transcripts in counteracting different cellular processes (e.g. tumor progression).
Specifically, the identification of a molecular mechanism controlling exosomal sorting of specific miRNAs paves the way for the design of chimeric miRNAs that specifically impact the exosomal informational content. We hypothesize that the engineering of specific miRNAs to be loaded into exosomes will magnify their delivery and therefore their biological function.
The experimental design is drawn to: i) provide proof of concept of the transferability in the development of novel therapeutic RNA-based strategies; ii) expand basic current knowledge in the field, therefore pinpointing more therapeutical tools and targets and iii) validate the results obtained to challenge a general value for the ncRNA-based approach.
The expected results include:
i)In vivo exosome-mediated delivery of antifibrotic chimeric miRNAs.
ii)Further exosome-sorting machinery characterization to expand the possibility of a exosomal miRNAs content control.
We expect to extend the basic knowledge on the molecular machinery controlling miRNA sorting in exosome and validate the possibility to in vivo specifically sort in exosome chimeric miRNAs embedding the hEXO motif.
The project is divided into two tasks, the first focusing on the attempt to transfer the acquired knowledge into innovative therapeutic strategies and the second one focusing on the extension of the basic studies of the described mechanisms to identify new molecular players:
Task 1: exosome-mediated delivery of antifibrotic miRNAs
We plan to engineer antifibrotic miRNAs by means of the embedment of a hEXO motif in their extra seed sequences to be loaded into hepatocyte exosomes. These chimeras will be tested for the correct sorting and their functionality both in vitro and in vivo. It¿s expected to set up a strategy of efficient in vivo delivery of miRNAs to ameliorate hepatic fibrosis.
We want to stress that the efficacy of this tool on fibrotic cells in vitro by targeting of miRNA29s, could represent the first step of the use of this method also for other miRNAs with the aim to modulate different patho-physiological processes (e.g. tumor progression).
Task2: exosome-sorting machinery characterization
These are independent tasks and potential pitfalls in one will not impede the other.
While our first evidence demonstrated that specific proteins may govern miRNA sorting by recognizing and binding of specific sequences in the RNAs, it is conceivable that a loading ribonucleoprotein machinery might involve several molecular players, each governing specific class of miRNAs. Further studies are here proposed to approach the systematic characterization of the ribonucleoprotein machinery, which might include tissue-specific cellular factors, that governs the exosome-mediated pivotal mechanism of cell-to-cell communication.
With respect to the experimental plan, the feasibility is guaranteed by the fact that all the proposed in vitro procedures are currently in use in the lab. We here like to stress that skills of our group include all the techniques of the below described experimental plane. In particular, we dispose of a proteomic unit equipped by: MALDI TOF/TOF (4800 MALDI-TOF/TOF; AB SCIEX); Nano High Pressure Liquid Chromatographer (UltiMate®3000 NanoLC-System; Dionex); Probot MALDI Spotter (Dionex); 2DE First dimension apparatus (Ettan IPGphor III; GE Healthcare); Spot-picker (Genomic Solutions ProPic);Typhoon scanner 9400 (GE Healthcare). With respect to the in vivo experiments, we rely on the expertise and facilities of the partner Giulia Piaggio, Department of Research, Diagnosis and Innovative Technologies, Translational Research Area National Cancer Institute Regina Elena.