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Human Exosomes: An Ideal Vehicle for Delivery of Therapeutic RNAs to Cells and Organs

Alexander KapustinAssociate Principal Scientist,

AstraZeneca

Hadi ValadiAssociate Professor, Sahlgrenska Academy,

Institute of Medicine, University of Gothenburg

Human Exosomes: An Ideal Vehicle for Delivery of

Therapeutic RNAs to Cells and Organs

Hadi Valadi, PhD, Associate [email protected]

Institute of Medicine,University of Gothenburg, Sweden 16

mailto:[email protected]

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RNA as a therapy molecule has a wide range of uses:

Treatment of monogenic diseases - there are over 10,000 different such disease, e.g. Huntington, Hemophilia, and cystic fibrosis.

Treatment of complex diseases, e.g. cancer, myocardial infarction

Create vaccines, for example cancer vaccine

RNA as a Therapy Molecule

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$92 millionRNA transport

$240 million

mRNA therapy

Investering siRNA therapeutics $ 1,1bn

Moderna mRNA therapeutic2012: $40 million2013: $24.6 million2013: $240 million (AstraZeneca, mRNA against cardiovascular, metabolic and renal diseases)2013: $110 million2014: $125 million2014: $100 million2014: $25 million2015: $950 million2016: $200 million (Merck, mRNA-basedpersonalized cancer vaccines)2016: $60 million (Vertex, against cystic fibrosis)2016: $125 million (BARDA, Zika mRNA)


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mRNA

Recipient Cells

mRNA

Vehicle (carrier)?

mRNA

protein

mRNAend up in

cytosol

Delivery

Protein production

Patient

A vehicle is required to transport therapeutic RNAs (siRNA, mRNA) to cells

Cytosol

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Nucleus

Therapeutic RNA

Vehicles for Delivery of Therapy RNAs

These vehicles (carriers) have not proven successful in clinical trials

Other Vehicles (carriers):

- Virus

- Lipid-based nano-particles

Activates the patient's immune system

Difficult to target to specific cell type or organ

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Vehicles for Delivery of Therapy RNAs

Need for a safe (non-immunogenic and non-

toxic) and efficient RNA/ DNA vehicle

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The body's own tool for

transporting therapeutic

substances (RNAs and proteins)

to cells and organs.

Exosomes from a patient do

not activate the patient's own

immune system (non

immunogenic)

Human exosomes, the body’s own vehicle to transport therapeutic RNA to cells

A Safe Carrier (Vehicle) for Transport of Therapeutic RNA

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What are exosome vesicles?

Exosomes - An Overview

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Lipid rafts:Flotillin-1

Exosome cytosolic proteins

Metabolism: GAPDH, PK, Enolase,P-Glycerate kinase, Peroxiredoin-1

Chaperones: HSP70, HSP90, Cyclophilin A

Membrane Trafficking: Rabproteins, Annexins, RAP-1β, Rho-GDI

Cytoskeleton: Actin, Tubulin, Myosin, Cofilin-1

Transmembranemolecules:

CD13, CD26, ATPase channels

Adhesion molecules:Tetraspanin,

Integrins, MFGE8

MHCIIMHCI

Exosomes, a population of extracellular vesicles

secreted by almost every cell type studied so far

30-150 nm in size

Endocytic origin

Secreted from many different cell types.

Different content depending on their cell origin.

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Early

endosomes

Cell

membrane

Exosome Production:

Particles

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Early

endosomes

Multivesicular

bodies (MVBs)

Form

intraluminal

vesicles of MVBs

Cell

membrane

Particles

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Autophagy

degradation pathway

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Multivesicular

bodies (MVBs)

Cell

membrane

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Multivesicular

bodies (MVBs)

Cell

membrane

Extracellular space

Exosome

vesicles

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First mentioned in the early 80s during maturation of sheep reticulocytes.

Pan, B.-T., and Johnstone, R.M. (1983). Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: Selective externalization of the receptor. Cell. 1983;33(3):967-78

MVBs

exosomes

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• Only blood Cells

• Only stem cells

• Only epithelial cells

• Just bacteria

• All cells studied so far

Which cells produce exosomes, or exosome like vesicles?

Audience Challenge QuestionANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT

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Exosome Producing Cells

Almost every cell type studied so far:

• Dendritic cells

• B cells

• T cells

• Mast cells

• Epithelial cells

• Stem cells

• Etc.

Presence of Exosomes

• Blood

• Human breast milk

• Saliva

• Urine

• and other body fluids

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Mouse mast cells exosomes Human mast cells exosomes

100 nm 100 nm

CD63-PE CD63-PE

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Mast Cells Exosomes

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Cut, purification,

and trypsin treatment

Nanoflow LC-MS/MS

> than 150 different

proteins were identified

Function Proteins

Chaperones endoplasmin, heat shock proteins (hsc

70, hsp 84, hsp 86)

Cytoskeleton actins (a, b)

Enzymes carboxypeptidase A, synthetase

Membrane

proteins

annexines (3, 5, 6, 8, 11), CD43, CD54,

CD63, CD97, integrines (a, b)

Protein synthesis ribosomal proteins (20S, 40S, 60S)

Signalling 14-3-3 protein

T cell stimulation MHCI

Total exosomal

proteins

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Proteomic Analysis

Total exosome protein identification showed the

presence of several RNA associated proteins in exosomes

Do exosomes contain

RNA or DNA ???

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Proteomic Analysis

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Detection of RNA in Exosomes

28 S

18 S

28 S

18 S

28 S18 S

Bioanalyzer results of cellular and exosomal RNA

Cel

lula

r R

NA

Exo

som

al R

NA

35

Exosomes Contain MicroRNA

• hsa-miR-765

• miRPlus_28993

• hsa-miR-122

• hsa-miR-483-5p

• hsa-miR-296-5p

• hsa-miR-96*

• hsa-miR-628-3p

• hsa-miR-193a-

• hsa-miR-518c*

• hsa-miR-637

• hsa-miR-492

• hsa-miR-371-5p

• hsa-miR-198

• hsa-miR-658

• hsa-miR-210

• miRPlus_17921

• hsa-miR-423-5p

• hsa-miR-611

microRNA-chip analysis revealed presence of 121 microRNAs in exosomes.

The expression of many of which were unique for the exosomes.

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Cel

lula

r m

RN

A

Cellular mRNA

Exo

som

al m

RN

A

Exosomal mRNA

Cellular mRNA

Exo

som

al m

RN

AEx

oso

mal

mR

NA

Exosomal mRNA c e c e c e c e

270 mRNA

37

Exosomes Contain mRNA

Uptake?Exosomes

3H RNA

0

5

10

15

20

25

30

P=0.02

P=0.02

P=0.01

Fold

ch

ange

(n

orm

alis

ed t

o t

=0)

Recipient cells: CD34 MC/9 HMC-1

Exosome donor cells Exosome recipient cells

38

Exosomes Can Transport RNAs between Cells

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• Only RNAs, such as mRNA and microRNA

• Just proteins

• Only lipid molecules

• A cargo of different components, e.g. RNAs, proteins and lipids

• Only small molecules

Which molecules or components can exosomes contain, or transport between cells?

Audience Challenge QuestionANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT

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RNA

RNA

RNA

RNA

Cells Cells

exosomer

RNA

RNA

RNA

Exosomal Shuttle RNA(esRNA)

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Transport RNA Between Cells via Exosomes

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To Use Exosomes as Carrier of Therapy RNA

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We hypothesize that:

It is possible to introduce exogenous RNAs into exosomes

Exosomes can deliver the functional RNA to cells in vitro & in vivo

Collaboration:

Exosome-based therapy

Patient

43

Insertion of Therapeutic RNA into Exosomes, and their use as ‘Biological RNA-Vehicle’

Cells Recipient cell

Nucleus

Cytosol

RNA

RNA

Exosomes

RNA

44

A Method is Needed to Insert Therapeutic RNAs into Exosomes

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Manuscript:

45

Preliminary Results:

Production of a human protein in mice (pg/ ml)

We hypothesized that:

Specific RNA binding proteins (RBPs)

transport some RNA (esRNA) from cytosol

into exosomes before they are secreted

into the extracellular environment

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Another Approach; using RNA-Binding Proteins (RBPs) to Introduce Exogenous RNA into Exosomes

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And it is possible to bind RNA

(modified RNA) to exosomal RBPsRBPs

47

The Mechanism by which RNA is Packaged into the Exosomes???

Statello L. et al. PLoS One. 2018 Apr 24;13(4).

48

We Therefore Searched for RBPs in Exosomes

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RNA-biotin

RNA-biotin-streptavidin

RBPs-RNA-biotin-streptavidin

RBPs

49

Identification of RBPs in Exosomes

Silencing of 6 Different Genes: HSP90AB1, XPO5, hnRNPH1, hnRNPM, hnRNPA2B1, and Major vault protein (MVP)


50

RNA-Binding Proteins (RBPs) are Involved in the Packaging of RNA into the Exosomes

Major vault protein (MVP)

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Specific RBPs are important for the

transport of RNA into exosomes during

biosynthesis of these vesicles

51

The Mechanism by which RNA is Packaged into the Exosomes


Exosomes are a population of EVs, secreted by almost all human cell types

Their molecular compartments originate partly from endosomes/ cell endocytosis

They contain a substantial amount of RNA (both mRNA and miRNA)

The RNA content of exosomes is complexed with RBPs

Exosomes can deliver a cargo of RNA and protein between cells

We hypothesize that:

It is possible to introduce exogenous RNAs into exosomes

Exosomes can deliver the functional RNA to cells in vitro and in vivo

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Summary

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Thank you!

University of Gothenburg, Sweden:Maugeri M, Nawaz M, Statello L, Wahlgren J, Papadimitriou A, Garre E, Na M, Camponeschi A, Sundqvist M, Mårtensson-Bopp IL, Jin T, Sunnerhagen P, and Valadi H

Collaboration/Projects:

AstraZeneca R&D:Angerfors A, Skantze P, Tigue N, HölttäM, Johansson S, Lindqvist J, Kjellman T, Östman S, Collén A, and Lindfors L

University of Catania, Italy:Ragusa M, Di Pietro C, Purrello M

And big thanks to:

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AstraZeneca



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Paul Hodges



Marshall Brennan


Nature

Jake Yeston

Science

Michael Torrice


Lauren Wolf






AstraZeneca



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Celebrating 4 years & 40 Drug Discovery Webinars!http://bit.ly/acsDrugDiscoveryArchive

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2014 2015 2016 2017

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59Find out more about the ACS MEDI Division! www.acsmedchem.org

Join the Division Today!

For $25 ($10 for students), You Will Receive:

• A free digital copy of our annual medicinal chemistry review volume (over 600 pages, $160 retail price)

• Abstracts of MEDI programming at national meetings

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Be a featured fan on an upcoming webinar! Write to us @ [email protected] 60

“It was encouraging to hear directly from Elizabeth de Lange that we need to face complexity and that reductionist approaches will not bring us further. A very interesting and enjoyable ACS Webinar.”

J.A. KellyACS member 24 years strong!


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@AmerChemSociety


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Benefits of ACS Membership


Chemical & Engineering News (C&EN) The preeminent weekly digital and print news source.

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NEW! ACS Career Navigator Your source for leadership development, professional education, career services, and much more.

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ACS Webinars does not endorse any products or services. The views expressed in this presentation are those of the presenter and do not necessarily reflect the views or policies of the American Chemical Society.

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Lauren Wolf


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