Extracellular Vesicles and MicroRNA: Definitions, Discovery, Clinical Potential and Open Questions

Extracellular Vesicles
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Reflecting on the discovery, open questions and potential applications of microRNA, and microRNA derived from extracellular vesicles.

Definitions of exosomes, extracellular vesicles and EV-microRNA

Extracellular vesicle microRNAs (EV-miRNAs) are among the range of molecular cargo contained in extracellular vesicles (EVs), which are a cell-derived, heterogenous population of phospholipid bilayer-enclosed particles.1 Among EVs are a population known as exosomes, which are defined by their biogenesis; upon fusing with the plasma membrane, multivesicular bodies release their intraluminal vesicles into the extracellular space. The released vesicles are known as exosomes, which are of particular interest for their role in cellular processes – including those involving miRNA. Exosomes have a typical diameter reported to be in the range of 40-150 nm.2 As it is difficult to distinguish exosomes from other similarly sized particles, exosomes are commonly referred to using the broader term of ‘EVs’.3

miRNAs are short, single-stranded molecules of RNA comprised of approximately 22 nucleotides in length.4 miRNAs influence a wide variety of biological processes through complex and dynamic mechanisms. As reviewed by O’Brien et al., miRNA exert their effects largely by inhibiting the production of proteins from mRNAs by binding to target mRNAs and either causing their degradation or by inhibiting their translation by the ribosomes.4 Such inhibition is known as gene silencing, and is mediated by associating with the multiprotein RNA-induced silencing complex.5 Several examples of miRNA-mediated upregulation of gene expression have also been reported in specific settings.6,7,8

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Extracellular vesicles and microRNAs share a slow rise to fame

It is now widely accepted that miRNA have important functional roles; however, miRNA were for a long time, largely overlooked. While the first example of a miRNA was initially identified in the nematode Caenorhabditis elegans in 1993,9 the next report of miRNA identification did not occur until the year 2000 – again in C. elegans.10 In the same year, a conserved, regulatory RNA was identified across a wide range of animal species, sparking a new era of research.11,12

Victor Ambros, co-author of the original 1993 paper, reflected on these developments in an insightful commentary:13

“After reading their paper in the autumn of 2000,12 I had to set aside 10 minutes to stare out the window and reorganize my view of the universe. Now we knew that lin-4 and let-7 RNAs were members of an evolutionarily ancient class of regulatory molecules, and so it was finally obvious that there must be other RNAs like them yet to be discovered in animals.”

These unexpected findings carried huge implications and helped bring the idea of ‘junk DNA’ (DNA which does not code for proteins) into question.14

The literature is now full of studies demonstrating associations between the abundance of specific miRNAs and certain conditions, highlighting their potential as biomarkers for everything from cancer15 to infectious disease.16  

Similarly, extracellular vesicles have a history of being underappreciated; initially, EVs were only recognised for their roles in waste removal.17 While waste removal is an important cellular function in itself, EV function is far more complex; EVs are now considered to be valuable intercellular messengers that transfer cargo between cells, including proteins,18,19,20 lipids21, DNA fragments22, messenger RNA (mRNA) and miRNA.23,24 Understanding the functional relevance – and potential clinical applications – of cargo transfer now represents significant opportunities for biomedical R&D.  

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Clinical utility of microRNA: under investigation

Before considering the potential clinical utility of EV-miRNA, it is worth considering the progress that has been made around developing and evaluating miRNA-based applications alone. Given miRNA can inhibit the expression of multiple target genes25, a number of clinically relevant questions have been raised:26  

  • Could miRNA be leveraged as a therapeutic payload?  
  • Could synthetic miRNAs be developed to downregulate genes of interest?  
  • Could anti-miRNAs be developed to target miRNA and upregulate gene expression?
  • What delivery methods might be appropriate here? Liposomes? Viral vectors? Exosomes? Other nanoparticles?

The applications of miRNAs also cannot be discussed without considering small interfering RNA (siRNA) which are another naturally occurring class of molecule. Like miRNAs, siRNA are short RNA duplexes that target mRNA(s) to produce a gene silencing effect.27 However, they have distinct differences related to their structure and mechanisms of action. One key difference is that while miRNA bind with partial complementarity to mRNA (allowing them to target multiple mRNA), siRNA are fully complementary to a single mRNA target.27

The therapeutic potential of miRNAs and siRNAs has been reviewed extensively28,29,30,31 and explored in preclinical studies32,33 and clinical trials:  

  • NCT02369198 involved a synthetic molecule designed to mimic a miRNA, encapsulated in an “EDV (EnGeneIC Delivery Vehicle)”, i.e., a delivery vehicle described as non-living bacterial minicells (nanoparticles). The novel chemotherapeutic drug was targeted to tumours via surface-attached bispecific proteins and was administered to patients with recurrent glioblastoma. Overall, the therapy was reported to be well tolerated; the Phase I study was completed and a recommended dose for a Phase II study was identified.34
  • NCT00882180 was a Phase I trial aimed at evaluating the safety, tolerability, pharmacokinetics and pharmacodynamics of an siRNA-based therapeutic formulated in lipid nanoparticles, targeted to VEGF and kinesin spindle protein (KSP) in patients with cancer. Several lines of evidence provide proof-of-concept for RNA interference as an approach for cancer treatment.35
  • NCT01829971 (Terminated): involved the administration of liposomal injections containing a miRNA called MRX34 to patients with primary liver cancer or other selected solid tumours or hematologic malignancies; unfortunately, the Phase I trial was halted following the occurrence of multiple immune-related serious adverse events.  
  • NCT00938574 and NCT01808638: Phase I and II studies aimed at evaluating a siRNA formulation given as a single and repeated intravenous infusion either on its own, or in combination with a standard chemotherapeutic, respectively, in patients with advanced solid tumours.36
  • NCT00496821: Established a proof-of-concept for an siRNA-based therapy directed against respiratory syncytial virus.37

The use of miRNAs for therapeutic purposes is an exciting yet ambitious endeavour; gene regulation is dynamic and complex, and research efforts have not yet fully translated to the clinical setting. In contrast to the handful of clinical trials that are directly evaluating miRNA-based therapeutic applications in cancer medicine, an overwhelming number are focused on evaluating miRNA-based diagnostic applications.31

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The clinical utility of EV-miRNA

Clearly, significant resources are being directed at understanding miRNA functions in vivo and evaluating miRNA-based applications. In this context, EVs add several new dimensions. EVs enriched with miRNA signatures indicative of the origin cell may serve as biomarker sources that provide far greater specificity than can be obtained through whole plasma analysis of miRNA.38 Furthermore, miRNA contained within EVs are protected from degradation in the circulation.  

To realise the diagnostic potential of EV-miRNA, a number of technical challenges must first be addressed. For example: how can EV miRNA be isolated and analysed in a standardised way? How might quantitative EV-miRNA data be normalised to provide clinically meaningful information to individuals? How can EV-miRNA diagnostic tests be refined to a sufficient level of sensitivity and specificity for clinical use?39

On a more basic level, there are also outstanding questions around the biology of EVs and their association with miRNA. For instance: to what extent are miRNAs associated with EVs? Does every EV contain miRNA? Does miRNA exist in EVs only within protein complexes?40 Are all EV-associated miRNAs contained within the lumen of EVs, or do surface miRNA exist also – and to what extent?41 What sort of engineering might be required to generate EVs with effective therapeutic cargo?42 Are EV-miRNAs bound to proteins from the silencing complex in EVs, or are they present as their hairpin precursors, as duplexes, bound to mRNA – or do they exist as free, mature miRNAs? These questions have been addressed in part, however the picture is incomplete. Furthermore, EV-miRNA loading into EVs has even been presented in some studies to be a passive43 and rare44 event, sparking debate among EV researchers45 that will likely continue for years to come.  

References

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  45. Extracellular Vesicle Club. Extracellular Vesicle microRNAs: Wimps or Warriors? https://www.youtube.com/watch?v=AgU4sM34AX8

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