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Graner. Extracell Vesicles Circ Nucleic Acids 2020;1:3-19 I http://dx.doi.org/10.20517/evcna.2020.08 Page 5

RFA-OD-20-018, seeking approaches to detect, test, validate, and implement testing for SARS-CoV-2 in the
COVID-19 pandemic. She ended with a list of exosome/EV-related funding announcements across NIH
Institutes, and commented on the future of vesicles and exRNA in terms of basic biology and translational
applications.

The next 2 sessions focused on exosome/EV cargo loading and cargo unloading. Session Chairs were
Juan Pablo Tosar (Universidad de la República, Uruguay) and Andrew Leidal (University of California
San Francisco, US). Alissa Weaver (Vanderbilt University, US) addressed a controversy in the EV field:
is EV-associated Argonaute an artifact or is it selected cargo? She noted that extracellular RNA in blood
is prevalent in non-vesicular forms, likely released from necrotic cells, and largely held by RNA-binding
proteins (RBPs), including Argonaute 2 (AGO2). Vesicular RNA in blood is less abundant, but is a product
of active secretion. These forms may represent different functional capacities, as well. She noted that different
cell status (mutations, signaling) result in different RNAs and RBPs that are expressed and localized in cells,
and differentially segregated into vesicles. She provided compelling evidence that serum (such as fetal bovine
serum) is a source of non-vesicular AGO2 and microRNAs, which further complicates the assessments
of AGO2 and miRNAs from cells grown in serum-containing media. Thus, the cell state, mutational and
signaling status, and growth conditions all contribute to AGO2 and likely, other RBPs, and their bound
RNAs in terms of vesicular or non-vesicular entities.

Leonid Margolis (NIH, National Institute of Child Health and Human Development, US) followed next
with a presentation on EV-associated cytokines and their implications for cell-cell communications. This
topic highlights broad questions of how exosomes and other EVs are addressed to particular cells, and how
the EV cargo affects recipient cells. EVs may play roles in protecting contents and applying appropriate
‘shipping addresses’. Leonid pointed out that cytokines are encapsulated within EVs, and are also present
on EV surfaces. These operate as systems, and that the cytokine surface vs. lumenal localizations are not
independent, but reflect changes in parental cell states. Differing soluble vs. cytokine-EV states are evident
in blood of patients experiencing post-radiation fatigue syndrome and in a form of myocardial infarction,
among other pathologic settings. EV-associated cytokines appear functional, and have an impact on recipient
cells due to their selective delivery onto cells with appropriate receptors. Improved understanding of cytokine
loading and targeting of such EVs could enlighten our concepts of cell-cell communication.

RNAs of various types are described as exosome/EV cargo, begging questions such as: how the RNAs are
loaded, are they transferred to recipient cells, and are the RNAs functional in the recipient cells. Olivier
de Jong (University Medical Center Utrecht, The Netherlands) reported studies in this area that employed
an innovative CRISPR-Cas9 system, which they called CRISPR operated stoplight system for functional
intercellular RNA exchange (CROSS-FIRE). It utilizes transfer of single guide RNAs (sgRNAs) that are
capable of driving reporter signals in recipient cells in a non-contact system, thus indicating the loading of
sgRNA into EVs in donor cells, and functional transfer to recipient cells measured by fluorescent readout.
This transfer worked with various donor and recipient cell types. The system uncovered genes involved in
endocytosis, extracellular matrix adhesion, and intracellular membrane trafficking that affected RNA transfer
via EVs (ITGB1, ROCK1, RAB5, RAB7). For translational development, the system appears more efficient at
functional RNA transfer than with lipid nanoparticle-based products, suggesting therapeutic uses of mRNA
or siRNA transfer.

In a display of EV cargo (or lack thereof) relevant to disease processes, Laura Ferraiuolo (University of
Sheffield, UK) introduced the neurodegenerative disease amyloid lateral sclerosis (ALS). The most frequent
mutation in the familial version of ALS (35%) and spontaneous ALS (11%) is in C9ORF72, which can lead to
several potential (and overlapping) pathological situations. One potential mechanism is that the C9ORF72
translation product may inappropriately sequester RNAs, thus altering cell function and cell crosstalk.