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Atlas (https://exrna-atlas.org). Their analysis led to the identification of six exRNA cargo types found
[65]
in multiple biofluids . While their findings suggest associations of cargo types with distinct carriers, it
also demonstrated that the heterogeneity of exRNA carriers and cargo types exceeds the capabilities of
current experimental methods to isolate and investigate specific carrier subpopulations and their cargo
[65]
in a reproducible way . The generation and optimization of methods to isolate high purity exRNA
subpopulations from biological samples, and, analyse the subsequent carrier exRNA contents, is a current
goal in the field.

EV targeting and cargo release
To be functional in the context of cell-cell signalling, an EV must also be able to find its physiological
target and release its cargo. But the question of how EVs target recipient cells can elicit a functional
cellular response is still unknown. The specificity of targeting EVs to recipient cells is thought to occur
through specific ligand-receptor interactions resulting in EV uptake. Mediators of these interactions
include tetraspanins, integrins, lipids, lectins, heparan sulphate proteoglycans, and extracellular matrix
components [60,66] . Once EVs are bound to the recipient cells, many different types of endocytotic processes
are known to mediate cellular uptake [13,60] . Membrane fusion is an alternative entry method in cancer
[67]
cells . However, different mechanisms of internalization have been described for different cell types, and
[66]
the mode of EV entry into target cells is thought to play a role in the functional effects . It is possible
that a population of EVs can simultaneously trigger a number of different methods of entry into a cell,
with the primary entry points depending on the cell type and EV cargo . Understanding the mechanism
[66]
of EV targeting and cargo release, and how this affects the functional fate of exRNA in recipient cells are
outstanding questions in exRNA biology.

ERCP
The NIH Common Fund-supported Extracellular RNA Communication Program (ERCP) was launched
in 2013 to accelerate progress in this new area of biomedical research. The overarching goal of the ERCP
has been to accelerate progress in the field exRNA biology and establish exRNA, and their carriers, as
mediators of intercellular communication. The first phase (stage 1) of the NIH Common Fund-supported
Extracellular RNA Communication Consortium (ERCC1) addressed five major challenges in the exRNA
[68]
field . The goals included: (1) to better understand the mechanisms of exRNA biogenesis, export and
secretion from the cell of origin; (2) to develop reference profiles for exRNA species from healthy human
biofluids; (3) to establish the utility of exRNA for biomarker development; (4) to establish the utility of
exRNA for therapeutic development; and (5) to develop community-wide resources for exRNA standards,
protocols, and data. The exRNA Portal (https://exrna.org/) is the central access point for ERCC resources
including descriptions of all ERCC projects, exRNA data and data standards, protocols, and computational
tools.

While significant advances were made during ERCC Stage 1, the exRNA field still faces many challenges, in
part due to both the inherent diversity of exRNA and the heterogeneity of exRNA carriers . In September
[61]
2019, the ExRNA Communication Program stage 2 (ERCC2) commenced to tackle the complexity of
exRNA molecules and the diverse array of exRNA carriers. ERCC2 researchers will develop tools to
efficiently and reproducibly isolate, identify, and analyse different carrier types and their exRNA cargos
and allow analysis of one carrier and its cargo at a time. The three major initiatives addressed in Stage 2 of
the ERCC include: (1) Improved Isolation and Analysis of exRNA-Carrier Subclasses; (2) Towards Single
Extracellular Vesicle (EV) Sorting, Isolation, and Analysis of Cargo; and (3) to serve as a community-
wide resource for exRNA standards, protocols, and data. The purpose of these initiatives is to further
characterize the cell or tissue from which their respective exRNAs originate and shed light on the diversity
of exRNAs carried by EVs. This will allow for a better understanding of the precise role of exRNAs as
signalling molecules for both physiological and pathophysiological processes, ultimately accelerating the
development of exRNAs for diagnostics.