The document summarizes work from the Extracellular RNA Communication Consortium (ERCC) on extracellular RNA (exRNA). It describes how exRNA profiles differ between biofluids like plasma, urine, saliva and cerebrospinal fluid (CSF). For example, over 60% of RNA in plasma is YRNA. It also highlights studies from the ERCC on the biogenesis of exRNAs, their potential as biomarkers for diseases like glioblastoma, and therapies using exosomes from stem cells which rescued mice from lethal liver failure.
exRNA Communication Mechanisms, Biomarkers, and Therapies
1. Extracellular RNA Communication:
Mechanisms, Biomarkers,
and Therapies
Friday 20 October, 2017
Scientific Outreach Coordinator
Extracellular RNA Communication Consortium
Roger P. Alexander
Senior Staff Scientist
Pacific Northwest Diabetes Research Institute
2. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
The Extracellular RNA Communication Consortium
2
OVERVIEW
• RNA was once thought to exist in a stable form only
inside cells, serving as an intermediate in the translation
from genes to proteins.
• However, recent research has indicated that RNAs can
play a role in a variety of complex cellular functions,
including newly discovered mechanisms of cell-to-cell
communication.
• RNA can be exported from cells in extracellular vesicles
(EVs) or bound to lipids or proteins, circulating through
the body and affecting distant cells and tissues. These
extracellular RNAs (exRNAs) may also be absorbed from
food, from the microbiome, or from the environment.
3. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
The Extracellular RNA Commuication Consortium
3
• The NIH Common Fund identified this new paradigm of intercellular
and inter-species information exchange as an important area of
inquiry, so in 2013 it launched the Extracellular RNA Communication
Consortium (ERCC).
• The goals of the ERCC are
– to discover fundamental biological principles about the
mechanisms of exRNA generation, secretion, and transport
– to investigate the potential for using exRNAs in the clinic as
• therapeutic molecules or
• biomarkers of disease
– to identify and develop a catalog of exRNAs found in normal
human body fluids
OVERVIEW
4. Outline
4
1. exRNA profiles in different biofluids
Examples of consortium work on
2. Biogenesis:
KRAS-dependent sorting of miRNA into exosomes
3. Biomarkers for glioblastoma
4. Therapy: Stem cell EVs rescue mice from liver failure
5. Consortium resources
– exRNA Portal
– exRNA Atlas
– Virtual Biorepository
OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCESOVERVIEW
5. Outline
5
1. exRNA profiles in different biofluids
Examples of consortium work on
2. Biogenesis:
KRAS-dependent sorting of miRNA into exosomes
3. Biomarkers for glioblastoma
4. Therapy: Stem cell EVs rescue mice from liver failure
5. Consortium resources
– exRNA Portal
– exRNA Atlas
– Virtual Biorepository
OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCESOVERVIEW
(Y-RNA is involved!)
6. Yeri, A. et al. Total extracellular small RNA profiles from plasma, saliva, and urine of healthy subjects. Sci. Rep. (2017) 7: 44061.
OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
RNA Profiles in different biofluids
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exRNA PROFILES
Plasma, urine, and saliva data are
from Yeri et al. 2017
CSF data is from the Jensen lab,
unpublished.
50-170 samples
of small RNA
purified from
whole biofluid
7. Yeri, A. et al. Total extracellular small RNA profiles from plasma, saliva, and urine of healthy subjects. Sci. Rep. (2017) 7: 44061.
OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
RNA Profiles in different biofluids
7
exRNA PROFILES
• On average, CSF and plasma have
much higher fractions of miRNA than
urine and saliva, about 25%.
• To date, most biomarker research
has focused on identifying miRNA
biomarkers of disease because of
their well-known function in negative
regulation of gene expression.
8. Yeri, A. et al. Total extracellular small RNA profiles from plasma, saliva, and urine of healthy subjects. Sci. Rep. (2017) 7: 44061.
OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
RNA Profiles in different biofluids
8
exRNA PROFILES
• Over 60% of the mappable RNA in
plasma is YRNA, almost entirely from
the 5’ end of RNY4. The human
genome contains 4 YRNA genes and
52 YRNA pseudogenes. YRNAs play
a role in chromatin interaction during
DNA replication, but their role as
exRNAs is not yet known.
9. Yeri, A. et al. Total extracellular small RNA profiles from plasma, saliva, and urine of healthy subjects. Sci. Rep. (2017) 7: 44061.
OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
RNA Profiles in different biofluids
9
exRNA PROFILES
• Over half of the RNAs in urine map
to multiple loci, mainly overlap piRNA
and tRNA fragments (tRFs).
• Otherwise, exRNA in urine consists
mainly of tRNA fragments.
• CSF also has a high fraction of
tRNA fragments.
10. Yeri, A. et al. Total extracellular small RNA profiles from plasma, saliva, and urine of healthy subjects. Sci. Rep. (2017) 7: 44061.
OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
RNA Profiles in different biofluids
10
exRNA PROFILES
• Saliva in particular includes a very
large fraction of exogenous RNA
from the oral microbiome (> 90%).
11. Kaczor-Urbanowicz, K.E.. et al.
Novel approaches for bioinformatic analysis of
salivary RNA sequencing data for development.
Bioinformatics (2017)
doi: 10.1093/bioinformatics/btx504
OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Exogenous RNA in saliva
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exRNA PROFILES
Saliva
12. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Exogenous RNA in saliva
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exRNA PROFILES
Saliva
Symposium at AADR 2018
(annual meeting of the
American Association
for Dental Research)
Salivaomics: Saliva Extracellular RNA (exRNA)
& the Saliva Proteome Wiki
13. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Biogenesis and delivery of exRNA
13
BIOGENESIS
Biogenesis, delivery, and function of extracellular RNA. Patton, J.G. et al.
J. Extracellular Vesicles (2015) 4:27494. doi: 10.3402/jev.v4.27494.
14. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
KRAS-dependent sorting of miRNA into EVs
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BIOGENESIS
KRAS-MEK Signaling Controls Ago2 Sorting into Exosomes. McKenzie, A.J. et al.
Cell Reports (2016) 15: 978–987.
When KRAS is hyper-active (MutDKO-1 cells), it inhibits Argonaute2
(Ago2) localization to multivesicular endosomes (MVEs).
MVE
marker
P body
marker
WT, low
KRAS
activity
mutant
hyper-
active
KRAS
15. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
KRAS-dependent sorting of miRNA into EVs
15
BIOGENESIS
KRAS-MEK Signaling Controls Ago2 Sorting into Exosomes. McKenzie, A.J. et al.
Cell Reports (2016) 15: 978–987.
When KRAS is hyper-active,
it initiates a MAP kinase cascade
that phosphorylates Ago-2 on S387
which inhibits Ago2 localization to
MVEs
and decreases secretion
in exosomes of Ago2
and miRNAs bound to it.
16. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Proving delivery of ncRNA to target cells
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DELIVERY
exRNA.org/About
For the complete story, watch James Patton’s seminar at
Hyper-active KRAS
in colorectal cancer cells
shunts miR-100 into EVs,
which transform target cells
when ingested.
17. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Glioblastoma EVs shape the tumor micro-environment
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BIOMARKERS
Skog, J. et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth
and provide diagnostic biomarkers. Nat. Cell Biol. (2008) 10: 1470-1476.
• Glioblastomas release copious numbers of
extracellular vesicles (EVs).
18. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
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• Those EVs are taken up by surrounding myeloid cells— microglia and macrophages.
• Normally functioning microglia are nurturers of self
but sentinels and warriors against other.
• Tumor EV uptake by microglia turns off their sentinel and warrior pathways and
activates nurturing of tumor cells.
BIOMARKERS
Glioblastoma EVs shape the tumor micro-environment
Skog, J. et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth
and provide diagnostic biomarkers. Nat. Cell Biol. (2008) 10: 1470-1476.
Glioblastoma cells grown for 3 days same, but supplemented with
glioblastoma microvesicles
19. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
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BIOMARKERS
• RNA and EVs from glioblastoma
show up in CSF
• Can CSF miRNA be used as
biomarkers for glioblastoma?
• ERCC researchers Fred Hochberg,
Ying Mao, Bob Carter, and Clark Chen
analyzed miRNA from 135 CSF
samples in 3 cohorts
using TaqMan OpenArray®
Human MicroRNA Panels
exRNA biomarkers for glioblastoma
Akers, J.C. et al. A cerebrospinal fluid microRNA signature as biomarker for glioblastoma. Oncotarget (2017) 8: 68769-68779.
20. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
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BIOMARKERS
Akers, J.C. et al. A cerebrospinal fluid microRNA signature as biomarker for glioblastoma. Oncotarget (2017) 8: 68769-68779.
• miRNAs with levels that differed between
GB and healthy CSF were identified using
the criteria
FDR < 0.2 and log(fold-change) > 2
exRNA biomarkers for glioblastoma
5 enriched in GB CSF
miR-21
miR-218
miR-193b
miR-331
miR-374a
4 depleted in GB CSF
miR-548c
miR-520f
miR-27b
miR-130b
• 24 miRNAs were selected
• LASSO regression down-selected to 9 miRNA:
21. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
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BIOMARKERS
Akers, J.C. et al. A cerebrospinal fluid microRNA signature as biomarker for glioblastoma. Oncotarget (2017) 8: 68769-68779.
exRNA biomarkers for glioblastoma
Performance of 9 miRNA classifier
(validated in 60 CSF samples from 2 cohorts)
22. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
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BIOMARKERS
exRNA biomarkers for glioblastoma
• miRNA profiles in lumbar and cisternal
CSF -- fluid collected from the spine or
the base of the neck, respectively --
are significantly different, which is
problematic, since cisternal CSF is
much more difficult to collect.
• On the other hand, they also found that RNAs
extracted from raw CSF had a similar profile and
diagnostic power as RNAs extracted from
vesicles after an initial EV purification step.
Akers, J.C. et al. A cerebrospinal fluid microRNA signature as biomarker for glioblastoma. Oncotarget (2017) 8: 68769-68779.
Figueroa, J.M. et al. Detection of wtEGFR Amplification and EGFRvIII Mutation in CSF-Derived Extracellular Vesicles of
Glioblastoma Patients. Neuro. Oncol. (2017) Advance online publication. doi: 10.1093/neuonc/nox085.
23. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Mice with lethal liver failure rescued
by extracellular vesicles from stem cells
23
• Co-administration of Tumor Necrosis Factor alpha (TNF-α)
and D-galactosamine (Dgal) results in liver failure,
characterized by
– tremulousness
– impaired ability to walk straight and rise from lying down
– loss of eyelash reflex
– eventual coma and death
• Liver failure can be rescued by administration of bone-
marrow-derived mesenchymal stem cells (MSCs).
• What about EVs from MSCs?
THERAPIES
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve survival from
lethal hepatic failure in mice. Haga H, Yan IK, Takahashi K, Matsuda A, Patel T.
Stem Cells Transl. Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.
24. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Mice with lethal liver failure rescued
by extracellular vesicles from stem cells
24
• x
THERAPIES
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve survival from
lethal hepatic failure in mice. Haga H, Yan IK, Takahashi K, Matsuda A, Patel T.
Stem Cells Transl. Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.
In mice with TNFα/Dgal-
induced liver failure,
mMSC-EVs are taken up
specifically by liver and spleen,
and not other organs.
IP
control
IP
liver injury
IV
control
IV
liver injury
25. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Mice with lethal liver failure rescued
by extracellular vesicles from stem cells
25
THERAPIES
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve survival from
lethal hepatic failure in mice. Haga H, Yan IK, Takahashi K, Matsuda A, Patel T.
Stem Cells Transl. Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.
n=8
n=6
n=6
n=8
n=7
n=8
n=8
n=7
n=7
n=6
n=4
n=4
n=4
57% survival at 24 hrs
37.5% survival at 24 hrs
26. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Mice with lethal liver failure rescued
by extracellular vesicles from stem cells
26
THERAPIES
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve survival from
lethal hepatic failure in mice. Haga H, Yan IK, Takahashi K, Matsuda A, Patel T.
Stem Cells Transl. Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.
lncRNA expression was
assessed by comparing RT-qPCR
in hMSC and hMSC-EV
...Turning an EV story into an exRNA story...
YRNA-1
27. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Mice with lethal liver failure rescued
by extracellular vesicles from stem cells
27
THERAPIES
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve
survival from lethal hepatic failure in mice.
Haga H, Yan IK, Takahashi K, Matsuda A, Patel T.
Stem Cells Transl. Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.
Caspase-3 (brown) is a marker of apoptosis.
Scale bar = 200 µM
YRNA-1
28. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Mice with lethal liver failure rescued
by extracellular vesicles from stem cells
28
THERAPIES
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve
survival from lethal hepatic failure in mice.
Haga H, Yan IK, Takahashi K, Matsuda A, Patel T.
Stem Cells Transl. Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.
Caspase-3 (brown) is a marker of apoptosis.
Scale bar = 200 µM
• Actinomycin-D induces apoptosis
Caspase 3/7 assay
of human hepatocytes
YRNA-1
29. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Mice with lethal liver failure rescued
by extracellular vesicles from stem cells
29
THERAPIES
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve
survival from lethal hepatic failure in mice.
Haga H, Yan IK, Takahashi K, Matsuda A, Patel T.
Stem Cells Transl. Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.
Caspase-3 (brown) is a marker of apoptosis.
Scale bar = 200 µM
• Actinomycin-D induces apoptosis
• hMSC-EVs reduce apoptosis
Caspase 3/7 assay
of human hepatocytes
YRNA-1
30. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Mice with lethal liver failure rescued
by extracellular vesicles from stem cells
30
THERAPIES
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve
survival from lethal hepatic failure in mice.
Haga H, Yan IK, Takahashi K, Matsuda A, Patel T.
Stem Cells Transl. Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.
Caspase-3 (brown) is a marker of apoptosis.
Scale bar = 200 µM
• Actinomycin-D induces apoptosis
• hMSC-EVs reduce apoptosis
• Loss of YRNA1 via siRNA knockdown
partially blocks the effectiveness of
hMSC-EVs
Caspase 3/7 assay
of human hepatocytes
YRNA-1
31. OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
Mice with lethal liver failure rescued
by extracellular vesicles from stem cells
31
THERAPIES
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve survival from
lethal hepatic failure in mice. Haga H, Yan IK, Takahashi K, Matsuda A, Patel T.
Stem Cells Transl. Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.
35. Upcoming Conferences
35
Newry, Maine August 19 - 24, 2018
Keystone Symposium on Exosomes/Microvesicles:
Heterogeneity, Biogenesis, Function, and Therapeutic
Developments Ÿ Breckenridge, Colorado Ÿ June 4-8, 2018
RNA Nanotechnology
Ventura, California Ÿ January 2019