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This ACS Webinar is co-produced with the ACS Division of Medicinal Chemistry and the American Association of Pharmaceutical Scientists13

www.acs.org/acswebinarsSlides available now and an invitation to view the recording will be sent when available.

A Nanomedicine Overview for mRNA Delivery: Innovative Methods Using Lipid NanoparticlesSession 3 of the 2018 Drug Design and Delivery Symposium

Marianna Yanez ArtetaSenior Scientist and Physical

Chemist Formulator, AstraZeneca

Dennis LeungSenior Scientific Manager,

Genentech

A Nanomedicine Overview for mRNA Delivery: Innovative Methods Using Lipid Nanoparticles

Marianna Yanez ArtetaACS Webinar: Drug Design and Delivery Series 2018 29 March 2018

28/03/2018

8

What will you learn in this webinar?

Nanomedicines for drug delivery

mRNA therapeutics: Promises, Challenges and Advances2

Lipid Nanoparticles (LNPs) for RNA delivery3

A (brief) background on Small Angle Scattering4

LNPs for mRNA delivery: Characterization and Performance5

Summary and Future Perspectives6

15

1

peptide drug

conjugatessmall molecules

~500

Why do we need nanomedicines?

Drugs are changing and their delivery requirements are changing.

16

Log Molecular weight

oligo-nucleotides

~5000

mRNA

~ 300, 000

peptides

~1400 siRNA

~13, 000

mAbs

~150,000

Figure courtesy of Marianne Ashford

53 42

28/03/2018

9

Nanomedicines for drug delivery

• Nanoparticles of drugs or biologics between 1-100 nm.

17

drug chemically

linked to a carrier

Dendrimers

Drug-

Polymer

Conjugates

Antibody Drug Conjugates

Liposomes

drug encapsulated

in a carrier

Inorganic

nanoparticles

Micelles

Polymeric

nanoparticles

Lipid

nanoparticles

Polymersomes

solid drug

nanoparticles

Figure courtesy of Marianne Ashford

Promises and Challenges of mRNA therapeutics

18

• Promises: Production of proteins

in vivo by administrating mRNA

• Challenges:

1. Crossing cell membrane for the

long negatively charged mRNA.

2. Enzymatic degradation of mRNA

before reaching target.

3. Finding a biocompatible vehicle.Schematic representation of the mechanism of

in vitro-transcribed mRNA translation and protein

replacement.

(Image courtesy of Kristina Friis)

28/03/2018

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• None

• 1

• 3

• 7

• 10

How many mRNA therapeutic treatments have been granted FDA approval?

Audience Challenge QuestionANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT

Clinical trials for RNA and delivery systems

20

Data taken from Kaczmarek et al. Genome Medicine 2017 and collated by Arpan Desai

Clinical trials involving RNA delivery by treatment and

clinical phase

Clinical trials involving RNA delivery by treatment and

delivery vehicle

28/03/2018

11

Lipid Nanoparticles (LNPs) for RNA delivery

LNPs: cationic ionizable lipid (CIL), cholesterol

(Chol), distearoylphosphatidylcholine (DSPC) and

a poly(ethylene glycol lipid).

21

Representation of LNP containing siRNA based

on molecular simulations

(Rozmanov et al. Faraday Discussions 2014)

CIL: Dlin-MC3-DMA

22

LNPs preparation approach: Using microfluidics

Easily produced with a

microfluidic mixer with a

high encapsulation

efficiency (> 90%).

Lipids (EtOH) mRNA (buffer)

Buffer exchange to

physiological conditions

Particle characterization

(size and encapsulation)

28/03/2018

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23

LNPs proposed mechanism of action for IV delivery to

hepatocytes

Akinc et al., (2010) Mol. Ther 18:1357-1364

• Alnylam Pharmaceuticals reported

successful on Phase III clinical trials

of Patisiran™, siRNA delivered

using LNPs for hATTR amyloidosis.

• Moderna has completed clinical

trials in Phase I/II for Zika

vaccination using LNPs.

24

Some examples of the use of LNPs in RNA

drug discovery

http://www.google.co.uk/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCOGUyuGYxscCFYNbFAodLDwDkQ&url=http://www.modernatx.com/&ei=bWbdVaGqN4O3Uaz4jIgJ&psig=AFQjCNFDDYCIZHJAeFom7kN9Y0-ZPS_wNA&ust=1440659388846821

http://www.google.co.uk/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCOGUyuGYxscCFYNbFAodLDwDkQ&url=http://www.modernatx.com/&ei=bWbdVaGqN4O3Uaz4jIgJ&psig=AFQjCNFDDYCIZHJAeFom7kN9Y0-ZPS_wNA&ust=1440659388846821

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25

Controlling the size of the LNPs

d (nm)

0 50 100 150 200 250

Num

ber

%

0

10

20

30

d (nm)

0 50 100 150 200 250

Num

ber

%

0

10

20

30

d (nm)

0 50 100 150 200 250

Num

ber

%

0

10

20

30

(a) (b) (c)

(d) (e) (f)

100 nm 100 nm 100 nm

89 nm62 nm43

nm

Standard approach:

CIL:Chol:DSPC:PEG-lipid

in a 50:40-x:10:x mole

ratio.

Size distributions: DLS number distribution vs. Cryo-TEM distribution

x = 3% x = 1.5% x = 0.5%

Yanez Arteta et al. PNAS Just accepted

t (h)

0 10 20 30 40 50Up

take

of

EP

O m

RN

A (

% o

f d

ose

)

0

20

40

60

80

100

t (h)

0 10 20 30 40 50Up

take

of

EP

O m

RN

A (

% o

f d

ose

)

0

20

40

60

80

100

26

In vitro uptake of LNPs of different size

89 nm62 nm43

nm 134 nm

LNP uptake in human adipocytes LNP uptake in iPSC derived hepatocytes

Tracking of LNPs

containing 3H labelled

DSPC.


28/03/2018

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t (h)

0 10 20 30 40 50Num

be

r of E

PO

per

mR

NA

do

se

d

0.0

0.1

0.2

0.3

0.4

0.5

0.6

27

Expression of EPO mRNA in vitro for LNPs of different size

t (h)

0 10 20 30 40 50Nu

mb

er

of

EP

O p

er

mR

NA

do

se

d

0.0

1.0

2.0

3.0

4.0

5.0

6.0

Protein expression in adipocytes Protein expression in hepatocytes

89 nm62

nm43

nm 134 nm

Why do LNPs of 62 nm

have a higher

transfection efficacy?


• I have measured small angle X-ray scattering (SAXS)

• I have measured small angle neutron scattering (SANS)

• I have vast experience in small angle scattering measurements

• I do not have experience with this technique

Have you performed small angle scattering experiments?

Audience Challenge QuestionANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT

28/03/2018

15

A (brief) background on Small Angle Scattering

Small angle scattering allows us to understand the shape and the

size of nanomedicines:

29

Is it

or

?

Sample

Neutron or X-raybeam

θki Detector

kf

q(Å-1)

0.0001 0.001 0.01 0.1 1

Inte

nsity (

a.u

.)

1e-6

1e-5

1e-4

1e-3

1e-2

1e-1

1e+0

1e+1

1e+2

1e+3

1e+4

Sphere modelCylinder modelData

q = ki-kf

θki

kf

Why do we use neutron scattering?

Selective deuteration of lipids and/or solvent produce different

scattering profiles.

30

Scattering Length Density (SLD, × 10-6 Å-2)

H2O D2O

-0.56 6.4

H2O

Dlin-KC2-DMA

DeuteratedCholesterol

DSPChead group D2O

DeuteratedDSPC

tail

7.7

DeuteratedDSPC

head group

DSPCtail

Cholesterol

RNAmatchpoint

p+

e-

p+ n0

e-

H1

1H2

1

ÅbH

51074.3 ÅbD

51067.6

b: Scattering length

28/03/2018

16

q(nm-1)

0.1 1 10

Inte

nsity

(a.u

.)

1e-5

1e-4

1e-3

1e-2

1e-1

1e+0

q(nm-1)

0.1 1 10

Inte

nsity

(a.u

.)

1e-7

1e-6

1e-5

1e-4

1e-3

1e-2

1e-1

mRNA-LNPs have a “structured core” with a 6 nm

correlation distance.

31

LNPs containing mRNA have a structured core

SAXS of mRNA LNPs vs. Empty LNPs

x = 1.5%, 43 nm

x = 3%, 45 nm

SAXS of mRNA LNPs of different sizes

• Inverted micellar phase?

(Literature)

• Onion?

• Wormlike micelles?


Why do we care about the structure of LNPs?

• LNP transfection efficacy is very low,

1-2% (Gilleron et al. (2013) Nat.

Biotech. 31:638-646)

• Which type of structures will facilitate

endosomal escape?

CAM2032: Fluid crystal © formulation for

prostate cancer Phase II.

32

Géral et al., (2013) Pharmaceutics 5:126-167

28/03/2018

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33

Location of lipids within the LNPs obtained by SANS

Radial distance (Å)

0 100 200 300 400

SL

D (

x1

0-6

Å-2

)

0

1

2

3

4

5

6

7

q(Å-1

)

0.001 0.01

Inte

nsity (

a.u

.)

1e-21e-11e+01e+11e+21e+31e+41e+51e+61e+71e+8

q(Å-1

)

0.01 0.1 1

Inte

nsity (

a.u

.)

1e-5

1e-4

1e-3

1e-2

1e-1

1e+0

1e+1

1e+2

1e+3

1e+4

q(Å-1

)

0.001 0.01 0.1

Inte

nsity (

a.u

.)

1e-41e-31e-21e-11e+01e+11e+21e+31e+41e+51e+61e+71e+8

(a) (b) (c)

(d) (e) (f)

AB

C

Solvent

A:

LNP core

d ~ 54 nm

B: Lipid monolayer (enriched

in DSPC): δ~2.4 nm

C: PEG2000 layer in mushroom

configuration: L~4 nm

A. LNP core:

• Cationic ionizable lipid (CIL)

• Cholesterol (CHOL)

• 24% water

• mRNA

D2O:

18%

27%

50%

68%

100%

SANS of LNPs with deuterated

DSPC and Chol in buffer with

different H2O/D2O ratio

SLD profiles corresponding to

the fits to the core-shell model

Schematic representation of

the lipid distribution in the

LNPs


34

Location of lipids within the LNPs: Comparison with

previous models

A:

LNP core

d ~ 54 nm

B: Lipid monolayer (enriched

in DSPC): δ~2.4 nm

C: PEG2000 layer in mushroom

configuration: L~4 nm

A. LNP core:

• Cationic ionizable lipid (CIL)

• Cholesterol (CHOL)

• 24% water

• mRNA


the lipid distribution in the

LNPs


Representation of an LNP containing siRNA :

CIL, Chol, DSPC and PEG-lipid. Based on

molecular simulations

(Rozmanov et al. Faraday Discussions 2014)

28/03/2018

18

Further exploration of the LNP core

Dialysis cassette:

CIL and CHOL in

EtOH

mRNA in water

Dialysis against

citrate buffer:ethanol

3:1

Dialysis cassette:

CIL and CHOL in

EtOH

mRNA in water

Dialysis against

PBS buffer

+/- +/-

35

Preparation buffer Storage buffer

Small angle x-ray scattering (SAXS) forempty and polyA LNP bulk phases (pH 3, 25% EtOH)

q (nm-1

)

0.1 1 10

Inte

nsity (

a.u

.)

0.1

1

10

100

SAXS of the core phase: citrate:ethanol 3:1 phase

cholesterol

monohydrate


a reversed hexagonal phase

structure. (Bilalov et al. Soft

Matter 2011)

q0

Reversed hexagonal phase (water or water/RNA rigid

cylinders):

• q1=√3*q0

• q2=√4*q0

Center-center distance a=6.2 nm

q1

q2

50 nm

20 nm

36

Freeze fracture micrograph

of the LNP core phase in

citrate:ethanol 3:1 phase


28/03/2018

19

Small angle x-ray scattering (SAXS) of mRNA LNPs vs empty LNPs

q (nm-1

)

0.1 1 10

Inte

nsity (

a.u

.)

1e-5

1e-4

1e-3

1e-2

1e-1

1e+0

1e+1

<d>N=45 nm (3 % DMPE-PEG)

<d>N=42 nm (1.5 % DMPE-PEG)

Small angle x-ray scattering (SAXS) forempty and polyA LNP bulk phases (pH 7.4)

q (nm-1

)

0.1 1 10

Inte

nsity (

a.u

.)

0.1

1

10

100

SAXS of the core phase: PBS buffer and comparison

with LNPs

q0

37 Yanez Arteta et al. PNAS Just accepted

50 nm

20 nm

Freeze fracture

micrograph of the LNP

core phase in PBS

Number average LNP diameter (nm)

40 60 80 100 120 140

Nu

mb

er

of

EP

O p

er

mR

NA

do

se

d (

at

48

h)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Number average LNP diameter (nm)

40 60 80 100 120 140Su

rfa

ce

are

a p

er

DS

PC

(n

m2/m

ole

cu

le)

0.4

0.6

0.8

1.0

1.2

1.4

1.6

DSPC in gel phase bilayer

38

Reprograming cell protein production by modifying

LNPs surface

Original

formulation

New formulation

(MC3:Chol ratio

constant)

Area per DSPC as a function of size

for different LNP formulations

Protein expression in adipocytes as a function of

the LNP size for different surface compositions


28/03/2018

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Summary and Future Perspectives

• LNPs are potential candidates for the delivery of mRNA.

• Small angle scattering provides a tool to characterize

nanomedicines.

• The transfection efficacy of LNPs containing mRNA is size and

surface composition dependent.

• Can these characterization methods lead towards the

optimization of other LNP formulations for mRNA delivery?

Lennart Lindfors

Tomas Kjellman

Xiaoqiu Wu

Aleksandra Dabkowska

Stefano Bartesaghi

Alexander Kvist

Simonetta Wallin

Special Thanks:

Annette Bak

Marianne Ashford

Arpan Desai

Kristina Friis

Noemi Szekely

Aurel Radulescu

Johan Bergenholtz

Marc Obiols-Rabasa (Lund University, SAXS

support measurements)

Jonny Eriksson (Uppsala University, cryo-TEM

measurements)

Acknowledgements

40

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Genentech

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

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2014 2015 2016 2017

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Genentech

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Chemical Entity and Biomolecule Scientific Program Tracks:

• Preclinical (including Discovery)• Bioanalytical• Clinical Pharmacology• Manufacturing & Bioprocessing• Formulation & Quality

YouTube video: https://www.youtube.com/watch?v=1DOxLBg0Ouw

Website: www.aapspharmsci360.org

46Find out more about the ACS MEDI Division! www.acsmedchem.org

Join the ACS Division of Medicinal Chemistry 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

• Access to student travel grants and fellowships


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Be a featured fan on an upcoming webinar! Write to us @ [email protected] 47

"ACS Webinars updates and encourages me as a recent graduate in diverse fields of pharmaceutical science. This webinar specifically helped come across members coming from relatable background and my desired career path. I hope to get in touch with them for guidance!”

How has benefited you?

Kirti ShettyMSc (Pharmacology), King’s College London

http://bit.ly/DDDS2video

48

youtube.com/acswebinars


28/03/2018

25

49

Benefits of ACS Membership


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

NEW! ACS SciFinderACS Members receive 25 complimentary SciFinder® research activities per year.

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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