Advances in RNAi Therapeutics Platform · 2019-12-14 · 3 Founded on the Bold Promise of Turning Nobel Prize Winning Science into a New Class of Medicine Alnylam Pharmaceuticals

1© 2018 Alnylam Pharmaceuticals, Inc.

22nd June, 2019

Advances in RNAi Therapeutics Platform

3rd International Conference on the Long and the Short of Non-Coding RNAs

Vasant Jadhav, PhD

2

Outline

• Introduction to RNAi Platform

• New Frontiers for RNAi Therapeutics: CNS and Ocular Delivery

• Mechanistic Understanding: Durability of RNAi Therapeutics

• RNAi Therapeutics Towards Non-Parenteral Dosing

3

Founded on the Bold Promise of Turning Nobel Prize Winning Science into a New Class of Medicine

Alnylam Pharmaceuticals

2018: Approval of first ever RNAi-based therapeutic by FDA and EMA

Discovery of RNAi in

mammalian cells

Elbashir et al., Nature,

2001;411:494-98

16-years later

2002: In vitro data by our scientific co-founders that started Alnylam

4 Structure adapted from Klosterman, P. S.; Shah, S. A.; Steitz, T. A Biochemistry (1999), 38, 14784-14792.

The Challenge

Making Drugs Out of siRNAs

21-23 base pairs long

dsRNA

2 bp (double) 3’ overhangs

Seed sequence

(residues 2-8)

Anti-sense or‘Guide’ strand

Sense strand

Characteristics

• M.W 12,000-14,000

• Size: 2 turns of helix

• 40 negative charges

• Hydrophilic

• Hydrated heavily

• ca. 5.5 nm X 2 nm

• Biostability

5

Lipid Nanoparticles (LNPs)

▪ siRNA (limited modifications)

in a multi-component lipid

formulation (LNP; ~85 nm)

▪ Targeted delivery to liver

▪ Intravenous (IV)

administration

Addressing the Delivery Challenge

GalNAc-siRNA Conjugates

▪ Single chemical entity

▪ Tri-GalNAc ligand conjugated

to sense strand of extensively

modified siRNA

▪ Targeted delivery to liver

▪ Subcutaneous (SC)

administration

Current Clinical

siRNAs

siRNA

Patisiran

Mechanisms for siRNA Delivery to Liver

6

GalNAc-siRNA Conjugates: SC-Administered Platform For Targeted Delivery To Hepatocytes

siRNAMetabolic stability

Intrinsic potency

Duration of effect

Safety

CMC

LigandReceptor affinity specificity

Metabolic stability

Safety

CMC

AsialoglycoproteinReceptor (ASGPR)

Highly expressed in hepatocytes

High turnover (recycling time ~15 min)

Conserved across species

7

Alnylam Clinical Development Pipeline

1 POC, proof of concept – defined as having demonstrated target gene knockdown and/or additional evidence of activity in clinical studies2 Approved in the U.S. for the polyneuropathy of hATTR amyloidosis in adults, and in the EU for the treatment of hATTR amyloidosis in adults with stage 1 or stage 2 polyneuropathy3 Includes marketing application submissions4 Cemdisiran is currently in Phase 2 development and pozelimab is currently in Phase 1 development; Alnylam and Regeneron are evaluating potential combinations of these two investigational therapeutics

As of May 2019

HUMAN

POC1

BREAKTHROUGH

DESIGNATIONEARLY STAGE

(IND or CTA Filed-Phase 2)

LATE STAGE (Phase 2-Phase 4)

REGISTRATION/

COMMERCIAL3

COMMERCIAL

RIGHTS

hATTR Amyloidosis2 ● Global

Givosiran Acute Hepatic Porphyria ● Global

PatisiranATTR Amyloidosis

Label Expansion ● Global

FitusiranHemophilia and Rare

Bleeding Disorders ● 15-30% royalties

Inclisiran Hypercholesterolemia ● Milestones & up to

20% royalties

Lumasiran Primary Hyperoxaluria Type 1 ● Global

Vutrisiran ATTR Amyloidosis ● Global

CemdisiranComplement-Mediated

Diseases ● 50-50

Cemdisiran/Pozelimab

Combo4

Complement-Mediated

Diseases ● Milestone/Royalty

ALN-AAT02 Alpha-1 Liver Disease ● Global

ALN-HBV02(VIR-2218)

Hepatitis B Virus Infection ● 50-50 option rights

post-Phase 2

ALN-AGT Hypertension ● Global

Focused in 4 Strategic Therapeutic Areas (STArs):Genetic Medicines Cardio-Metabolic Diseases

Hepatic Infectious Diseases CNS/Ocular Diseases

8

Outline

• Introduction: RNAi Platform




9

RNAi Therapeutics for CNS and Ocular Diseases

Enormous unmet medical need across the CNS and Ocular spaces

• Many dominantly inherited eye

diseases

• Many dominantly inherited

neurodegenerative diseases

10

Superior silencing to parent at 10-fold lower dose

SOD1 siRNA Conjugates Demonstrate Superior Silencing in Rat CNS

Lumbar

Thoracic

Cervical

Cerebellum

Frontal Cortex

Temporal Cortex

Control

SOD1 parent- 0.9 mg

SOD1 modified 0.9 mg



Cerebellum

Frontal

Cortex

Rest of brain

11

Single IT dose- 0.9 mg

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 1700.00

0.25

0.50

0.75

1.00

1.25

1.50

Time (Days)

Temporal Cortex

Durable Silencing - 0.9 mg

Messag

e R

em

ain

ing

Lumbar spinal cord

Cerebellum

Hippocampus

Frontal Cortex

Thoracic spinal cord

Cervical spinal cord

Up to 6 months of silencing in some regions of the CNS following a single dose

Robust Silencing Throughout the CNS

• Durable lowering across animals in most regions of the brain for up to 6 months

• Silencing in spine maintained close to NADIR through 6 months

• PD comparison in liver across species together with extended duration seen in rodents expected to support

infrequent dosing in human

12

siRNA vs ASO in hSOD1 (SOD1G93A) Rats

McCampbell et al, J Clin Invest. 2018;128(8):3558-3567

Study Purpose

• Head-to-head comparison of siRNA and ASO– siRNAs selected from mouse AAV-hsSOD1 screen

– ASO 1, based on McCampbell et al. (2018)◦ Demonstrated ~75% maximum silencing at 2 weeks in the same rat model

Study Design

• Single IT injection of 0.9 mg assayed at day 7– Same dose used for siRNA and ASO

• Single IT injection of 0.45 mg assayed at day 28

Day 7 or 28 collection after single IT dose

Study Day

Tissues collected

across spinal cord

and brain

0 7

IT injection siRNA

& ASO

Tissues collected

spinal cord

28

0.45 mg 28 day

0.9 mg 7 day

13

Improved hSOD1 mRNA reduction with siRNA compared to ASO in CNS at day 7


aC

SF

AS

O

siR

NA

AS

O

siR

NA

AS

O

siR

NA

AS

O

siR

NA

AS

O

siR

NA

AS

O

siR

NA

AS

O

siR

NA

AS

O

siR

NA

0

20

40

60

80

100

120

% h

SO

D1 m

essage r

em

ain

ing

rela

tive to a

CS

F

Lumbar Thoracic CervicalBrain

StemCerebellum Hippo

Frontal

CortexTemporal

Cortex

Greater silencing in all regions of the brain and spinal cord was observed using an

siRNA targeting hSOD1 in this model at day 7

Single IT dose of 0.9 mg

14

Improved hSOD1 mRNA reduction with siRNA compared to ASO in the spine at day 28


Greater silencing in all regions of the spinal cord were observed using an

siRNA targeting hSOD1 in this model at day 28

Single IT dose of 0.45 mg

aCSF ASO siRNA ASO siRNA ASO siRNA

0

20

40

60

80

100

120

% h

SO

D1 m

essage r

em

ain

ing

rela

tive to a

CS

F

Lumbar Thoracic Cervical

15

Ocular TTR Silencing by Differentially Modified siRNA Conjugates in Rat

After Single Intravitreal Injection

PB

S

Par t

ially M

od

ifie

d

ES

C

EH

op

tim

ized

0 .2 5

0 .5

1

2

4

8

1 6

3 2

6 4

1 2 8

2 5 6

% m

es

sa

ge

re

ma

inin

g

Rat TTR mRNA

Day 14, 50 mg siRNA conjugate

16

Dose Response and Duration of Activity of Ocular siRNA Conjugates in Mice After Single Intravitreal Injection

PB

S

1 u

g

7.5

ug

11.6

ug

0

5 0

1 0 0

1 5 0

O c u la r m T T R d o s e re s p o n s e

IV T D ay 13

% m

es

sa

ge

re

ma

inin

g

(re

lati

ve

to

PB

S)

PB

SD

28

D56

D84

D112

D135

0

5 0

1 0 0

1 5 0

O c u la r m T T R d u ra tio n 5 m o n th s

s in g le 1 5u g IV T in je c tion

% m

es

sa

ge

re

ma

inin

g

(re

lati

ve

to

PB

S)

Excellent duration observed for siRNA conjugates in eye

17

Current Ocular Design Shows Impressive Potency and Duration in NHP

P B S A D -2 9 1 8 4 5

0 .0 0 3 m g

A D -2 9 1 8 4 5

0 .0 3 m g

A D -2 9 1 8 4 5

0 .1 m g

A D -2 9 1 8 4 5

0 .3 m g

0

2 5

5 0

7 5

1 0 0

1 2 5

% T T R R e m a in in g (T T R A D -2 9 1 8 4 5 )

D a y 2 8 A q u e o u s H u m o r

% T

TR

Re

ma

inin

g

Re

lati

ve

to

PB

S C

on

tro

l

M in

% R e m a in in g

P B S A D -2 9 1 8 4 5

0 .0 0 3 m g

A D -2 9 1 8 4 5

0 .0 3 m g

A D -2 9 1 8 4 5

0 .1 m g

A D -2 9 1 8 4 5

0 .3 m g

0

2 5

5 0

7 5

1 0 0

1 2 5

% T T R R e m a in in g (T T R s iR N A d o s e d g ro u p s )


% T

TR

Re

ma

inin

g

Re

lati

ve

to

PB

S C

on

tro

l

M in

% R e m a in in g

P B S A D -2 9 1 8 4 5

0 .0 0 3 m g

A D -2 9 1 8 4 5

0 .0 3 m g

A D -2 9 1 8 4 5

0 .1 m g

A D -2 9 1 8 4 5

0 .3 m g

0

2 5

5 0

7 5

1 0 0

1 2 5



% T

TR

Re

ma

inin

g

Re

lati

ve

to

PB

S C

on

tro

l

M in

% R e m a in in g

P B S A D -2 9 1 8 4 5

0 .0 0 3 m g

A D -2 9 1 8 4 5

0 .0 3 m g

A D -2 9 1 8 4 5

0 .1 m g

A D -2 9 1 8 4 5

0 .3 m g

0

2 5

5 0

7 5

1 0 0

1 2 5



% T

TR

Re

ma

inin

g

Re

lati

ve

to

PB

S C

on

tro

l

M in

% R e m a in in gP B S A D -2 9 1 8 4 5

0 .0 0 3 m g

A D -2 9 1 8 4 5

0 .0 3 m g

A D -2 9 1 8 4 5

0 .1 m g

A D -2 9 1 8 4 5

0 .3 m g

0

2 5

5 0

7 5

1 0 0

1 2 5



% T

TR

Re

ma

inin

g

Re

lati

ve

to

PB

S C

on

tro

l

M in

% R e m a in in g

P B S A D -2 9 1 8 4 5

0 .0 0 3 m g

A D -2 9 1 8 4 5

0 .0 3 m g

A D -2 9 1 8 4 5

0 .1 m g

A D -2 9 1 8 4 5

0 .3 m g

0

2 5

5 0

7 5

1 0 0

1 2 5



% T

TR

Re

ma

inin

g

Re

lati

ve

to

PB

S C

on

tro

l

M in

% R e m a in in g

P B S A D -2 9 1 8 4 5

0 .0 0 3 m g

A D -2 9 1 8 4 5

0 .0 3 m g

A D -2 9 1 8 4 5

0 .1 m g

A D -2 9 1 8 4 5

0 .3 m g

0

2 5

5 0

7 5

1 0 0

1 2 5



% T

TR

Re

ma

inin

g

Re

lati

ve

to

PB

S C

on

tro

l

M in

% R e m a in in g

P B S A D -2 9 1 8 4 5

0 .0 0 3 m g

A D -2 9 1 8 4 5

0 .0 3 m g

A D -2 9 1 8 4 5

0 .1 m g

A D -2 9 1 8 4 5

0 .3 m g

0

2 5

5 0

7 5

1 0 0

1 2 5



% T

TR

Re

ma

inin

g

Re

lati

ve

to

PB

S C

on

tro

l

M in

% R e m a in in g

Excellent duration observed for siRNA conjugates in NHP eye

18

Alnylam-Regeneron Alliance*

* Alliance and equity agreements with Regeneron expected to close during Q2 2019

Landmark Alliance Focused on CNS & Ocular RNAi Therapeutics

• Partnership of two leading biopharmaceutical companies committed to innovation

‒ Alnylam R&D expertise and scientific excellence in RNAi therapeutics with emerging global commercial presence

‒ Regeneron scientific excellence, world-leading capabilities in human genetics, and industry-leading commercial presence in

ophthalmology and other large markets

• Broad, multi-product alliance across CNS, ocular, and select liver targets

‒ Both companies fully participate in value creation with 50-50 structure in CNS and select liver programs

‒ Milestone/royalty structure for ocular disease programs

• Accelerates Alnylam CNS and ocular programs, driving significant pipeline expansion

‒ Robust, highly durable, and widely distributed RNAi knockdown of key targets in CNS/ocular pre-clinical models

‒ Adds 1-2 new planned INDs/year toward CNS or ocular targets to previously planned 1-2 new INDs/year in liver beginning in 2020

• Significantly bolsters Alnylam balance sheet to >$2B pro forma for increased pipeline investment and future growth

19

Outline





20

Extended Duration of Activity by ESC Conjugates

Rela

tive

le

ve

ls o

f s

eru

m b

iom

ark

er

Human pharmacodynamic response* of two siRNAs with the same sequence, different chemistry

DaysSTC qD x 5, qWx5

Advanced ESC Single dose

STC: 500 mg (qDx5, qWx5)

Advanced ESC: 50 mg (single

dose)

*Phase 1 data in healthy volunteers from separate studies

2’-Fluoro 2’-OMe PS- Phosphorothioate

Advanced ESC: Enhanced Stability ChemistrySTC: Standard Template Chemistry

21

• Sustained release of conjugate from SC injection site to liver?

• Increased half-life of siRNA-loaded RISC?

• Continuous supply of siRNA from an intracellular depot?

Depot Effect Hypothesis for Conjugate Extended Duration of Effect

SC

Injection site

GalNAc-siRNA

conjugate

Circulation

Liver

Hepatocyte uptake, intracellular

trafficking and release

22

The SC Injection Site Is Not A Depot For GalNAc-siRNA - IV Dosing Of Potent

Compounds Shows Similar Profile

SC

Depot?

IV

No potential SC depot

vs

Nair et al., NAR, 2017

Increase

stability

23

In Vivo Duration Of Silencing In Mice Is Dependent On Delivery Modality

And Stability

• Unlike GalNAc-siRNA conjugates, LNP designed to promote efficient endosomal escape of siRNAs

• Doses selected to get similar level of KD and thus similar level of RISC loading expected

• Faster onset and recovery of activity with LNP

• Slower onset but substantially extended duration with GalNAc-conjugate

• Overall data suggests that RISC half-life alone can not explain the duration of activity for conjugates

24

Functional siRNA Released From Acidic Compartments Up To Three Weeks

Post-Dose

GalNAc-siRNA

8h, days

11 or 21

siRNA

release

GalNAc Endolytic

Peptide

0

10

20

30

40

50

60

70

0 10 20 30 40 50

Pe

rce

nt

Seru

m T

TR

(Re

l. t

o P

re-D

ose

)

Study Day

TTR KnockdownAdvanced ESC - 0.5 mg/kg

No Peptide

8h Peptide

D11 Peptide

D21 Peptide

siRNA

Lysopainter

25

Weeks After Conjugate Dosing, Ectopically Expressed Tagged Ago2 Continues

To Load siRNA

GalNAc-siRNA

7 days

FLAG-mAgo2 AAV

23 days

No continuous

loading

Continuous

loading

160

90

kD

*

0

0.1

0.2

0.3

0.4

0.5

An

tisen

se S

tran

d

(ng

/g)

Antisense RISC LoadingDay 30

26

Outline





27

Needle Free Delivery of GalNAc-siRNA via LungPoC Demonstrated in Mice Using Microsprayer®

Microsprayer® - A high pressure syringe for direct administration

of aerosol at the junction of trachea for delivery in lung

2

7

Microsprayer® developed by PennCentury

Systemic exposure of siRNA and LNA-antisense oligos by intra-tracheal dosing. Molecular Therapy 2011 19 (12), 2163–2168

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 7 14 21

No

rma

lize

d s

eru

m T

TR

le

ve

ls

Days Post Dose

Microsprayer® Mediated Dosing Achieves Comparable Potency and Duration of

Activity to SC Delivered ESC Conjugates in Mouse Liver

Saline

1 mg/kg

3 mg/kg

Presented at OTS 2015

SC Microsprayer®

Given the superior potency, metabolic stability and durable activity of GalNAc-siRNA conjugates, would they

also work via Oral Dosing- The least invasive method?

28

PoC for Oral Dosing of GalNAc-siRNA in Mice Delivered via Gavage Tube

The Laboratory Mouse (2nd Edition)

2012, Pages 709-725

The feeding tube is passed gently through the mouth and

pharynx into the esophagus to deposit solution in stomach

Day 0 145 7

Serum collection for biomarker analysis

21

Study design

▪ ESC siRNA +/- GalNAc

▪ Formulation containing permeation enhancer

▪ Single or 3 doses at Day 1, 2 and 5

Flexible Plastic Feeding TubesInstech’s plastic gavage tubes are

flexible to reduce trauma

29

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 5 10 15 20 25 30 35 40 45

Days

Serum biomarker levels post siRNA dosing in mice

Robust and Durable Activity Seen by Oral Dosing of GalNAc-siRNA in Mice

PBS

3 x 10 mg/kg GavageRela

tive

F1

2 leve

ls

1 x 0.75 mg/kg SC

Oral gavage

30

Dose Dependent Activity Seen by Oral Dosing of GalNAc-siRNA in Mice

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25

Re

lati

ve

Le

ve

ls o

f B

iom

ark

er

Days

3 X 10 mg/kg

3 X 3 mg/kg

10 mg/kg

3 mg/kg


Oral gavage

31

GalNAc Conjugation and Formulation are Important for siRNA Activity via

Oral Dosing

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 5 10 15 20 25

Re

lati

ve

bio

ma

rke

r le

ve

ls

Days


Without formulation

Unconjugated siRNA

3 x 10 mg/kg

With formulation

GalNAc-siRNA

3 X 10 mg/kg

Oral gavage

32

RNAi therapeutics emerging as high impact, transformational medicines

◦ ONPATTRO® as 1st RNAi therapeutic is now in market serving patients

◦ Multiple RNAi therapeutics are in advanced stages of clinical development

New frontiers for future expansion of RNAi therapeutics opportunity

◦ Delivery of RNAi therapeutics to CNS and eye achieved

◦ Our learnings in the liver apply!!

Preclinical data suggests durability of GalNAc-siRNAs likely from continuous supply of siRNA from intracellular depot

Achieved PoC for oral dosing of GalNAc-siRNA conjugates- the least invasive method of drug administration

◦ Convenience of conventional dosing for modern medicine

Summary

33

Acknowledgements

Our volunteers, patients and patient families

Thank you!

Participating volunteers, patients and their families

Alnylam colleagues: Research Department Early Development RNAi Platform

MGH: Dr. Brown lab

Advances in RNAi Therapeutics Platform · 2019-12-14 · 3 Founded on the Bold Promise of Turning Nobel Prize Winning Science into a New Class of Medicine Alnylam Pharmaceuticals

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