Pg. 1

Restoring Biological Harmony for Patients with Debilitating DiseaseRestoring Biological Harmony for Patients with Debilitating Disease

The miR-29b Mimic Remlarsen as an Anti-Fibrotic Therapeutic in the EyeCorrie Gallant-Behm, PhD, MQARS

AOPT 20190310

Pg. 2

Fibrosis is a Significant Cause of Blindness

▪ Corneal fibrosis is second leading cause of blindness (after cataracts) worldwide. Results from corneal damage beyond Bowman’s membrane:▪ Viral keratitis

▪ Bacterial keratitis

▪ Fungal keratitis

▪ Sterile keratitis, dry eye disease

▪ Physical damage to cornea (trauma, surgery, thermal and chemical burns)

▪ Retinal fibrosis is the final common pathophysiological denominator and a major contributor to vision loss from:▪ Age related macular degeneration

▪ Diabetic retinopathy

▪ Retinal detachment (proliferative vitreoretinopathy)

▪ Retinopathy of prematurity

▪ Neovascular glaucoma

Pg. 3

▪ Reduced Expression of miR-29 has been Implicated in the Development and Progression of a Wide Range of Fibrosis Indications

miR-29 is an Anti-Fibrotic miRNA

Ocular fibrosis:Corneal fibrosis

Fuch’s Endothelial Corneal Dystrophy

Glaucoma/trabeculectomy bleb

Retinal fibrosis

Age related macular degeneration

Diabetic retinopathy

Pulmonary fibrosis

Inflammatory bowel disease

Liver fibrosis

Cardiac fibrosis

Tendinopathies

Renal fibrosis

Cutaneous fibrosis

Osteoarthritis

Dupuytren’s contractures

miR-29TGF-b

+ ECMFibrosis

miR-29

agonist

TGF-b

+ ECMFibrosisX X

▪ miR-29 inhibits inflammation, TGF-b activity, EMT, fibroblast-to-myofibroblast transition and ECM synthesis

▪ miR-29 inhibits every step of the collagen fibrillogenesis pathway

miRagen preclinical and/or

clinical data + literature support

Literature support

Pg. 4

Remlarsen Impacts Multiple Fibrotic Processes

Remlarsen Corticosteroids (SOC)

Reduces inflammation ● ●

Accelerates healing ●

Reduces EMT ●

Reduces fibroblast activation ●

Reduces myofibroblasts ●

Inhibits collagen production ●

Pg. 5

The Eye is a Preferred Target Organ for microRNA Therapeutics

▪ Delivery to multiple compartments of the eye, and via multiple routes of administration including injection and topical drops

▪ Stability due to reduced nuclease concentration enables long duration of action and infrequent dosing

▪ Reduced liability for toxicities due to local administration with limited systemic exposure

▪ Remlarsen is a clinical stage miR-29b mimic that has been shown to downregulate the expression of multiple collagens and other ECM molecules in the skin in humans and to inhibit fibroplasia in iatrogenic cutaneous wounds in normal healthy volunteers. It has also been shown to regulate this same expression in in vivo animal models of pulmonary, hepatic, corneal and retinal fibrosis.

Pg. 6

Topically Applied Remlarsen Eye Drops Are Readily Taken Up by Damaged Corneas

▪ Distribution of drug to all layers of the cornea, including corneal endothelium, after drop administration▪ Animal model: Rat alkali burn

▪ Formulated in simple buffer with no additional stabilizers

▪ No penetration enhancement/nanoparticle formulation required

EpitheliumStroma

FITC-remlarsen

DAPI

Pg. 7

▪ Uptake of remlarsen in alkali burned cornea is high in the presence of corneal compromise and remains elevated for up to 28 days

Remlarsen Eye Drops Demonstrate Sustained Uptake in Alkali Burned Cornea

0

5

1 0

1 5

2 0

2 5

6 0

8 0

1 0 0

1 0 0 0

2 0 0 0

3 0 0 0

miR

-29

ex

pre

ss

ion

(fo

ld c

ha

ng

e v

s.

sa

lin

e)

S a lin e 1 2 4 7 1 0 1 4 2 1 2 8

D a y s a fte r re m la rs e n tre a tm e n t in it ia tio n

1717x

14x

4x

13x

1 .7x

58x 61x

11x

N=6 per group

Pg. 8

Remlarsen Eye Drops Accelerate Healing of Damaged Cornea

▪ Decreased inflammation and stromal damage

▪ Restored normal corneal architecture after alkali burn

No Burn

Burn

+

Saline

Burn

+

Remlarsen

Images taken Day 4 post-burn

Thin, friable epithelium

Infiltrating immune cells

Stromal damage

Epithelium nearly normal

Minimal inflammatory infiltrate

Stromal damage absent

Pg. 9

Remlarsen Accelerates Corneal Healing and Reduces Scarring

▪ Epithelium thickness restored to normal following remlarsen treatment

▪ Stromal thickness (scarring) reduced with remlarsen treatment

S a lin e R e m la r s e n

0

2 0

4 0

6 0

Ep

ith

eli

um

th

ick

ne

ss

(

M)

p < 0 .0 1

N = 1 2 N = 1 1

S a lin e R e m la r s e n

0

1 0 0

2 0 0

3 0 0

Str

om

al

thic

kn

es

s (

M)

p < 0 .0 5

N = 1 2 N = 1 1

Dotted line indicates normal corneal thickness

Day 14 after corneal burn

Epithelial thickness Stromal thickness

Pg. 10

Remlarsen Reduces Corneal Scarring

▪ Reduced corneal hazing, scarring▪ Increased corneal transparency

▪ Reduced a-SMA expression▪ Reduced EMT

▪ Reduced FMT

S a lin e R e m la r s e n

1

2

3

4

5

Ha

zin

g/s

ca

rrin

g s

co

re

(1

-5)

p = 0 .0 0 3

N = 1 2 N = 1 2

Burn + Saline

Burn + Remlarsen

Images taken Day 10 post-burn, 20x magnification

Double-masked observer determination

according to the following scoring system

(Scoring Day 10 post-burn):

1 = normal

2 = slight haze

3 = moderate haze

4 = severe haze + slight scar

5 = severe haze + overt scar

Pg. 11

▪ miR-29b PD biomarkers are repressed following twice daily remlarsen drop treatments

▪ Consistent in multiple studies

▪ Multiple collagens and pro-fibrotic factors are affected

▪ Remlarsen typifies pathway/network regulation by microRNAs

▪ Regulation of many genes concurrently leads to coordinated impact on pathology

Remlarsen Demonstrates Target Engagement in Cornea in vivo

S a lin e R e m la r s e n

0 .0

0 .5

1 .0

1 .5

T a r g e t E n g a g e m e n tm

RN

A E

xp

re

ss

ion

(Fo

ld C

ha

ng

e)

p < 0 .0 0 0 1

COL1A1

COL1A2

COL5A2

FN1

TGFB2

MMP2

Day 14 after corneal burn

N=6 per group

Pg. 12

Remlarsen is Well Taken Up by All Layers of the Retina After IVT Administration

▪ Distribution of remlarsen to all layers of the retina, including pigmented epithelium via intravitreal injection

▪ Formulated in simple buffer with no nanoparticles or exotic formulation required

Lens

Ganglion cell layer

Inner plexiform layer

Inner nuclear layer

Outer nuclear layer

Photoreceptor inner

segments

Photoreceptor outer

segments

Outer plexiform layer

DAPI

FITC-remlarsen 20x 40x

Photoreceptor outer

segments

RPE layer

Pg. 13

Remlarsen Demonstrates Favorable PK in the Retina

▪ Remlarsen retention in retina and back of the eye for > 1 week after single intravitreal injection; later time points not yet tested

▪ Supports infrequent dosing

S a l in e 2 4 h rs 4 8 h rs 4 d a y s 7 d a y s

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

m iR -2 9 b le v e ls in ra t re t in a

miR

NA

ex

pre

ss

ion

(fo

ld c

ha

ng

e)

* *

R e m la r s e n

S a l in e 2 4 h rs 4 8 h rs 4 d a y s 7 d a y s

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

m iR -2 9 b le v e ls in ra t R P E , c h o ro id & s c le ra

miR

NA

ex

pre

ss

ion

(fo

ld c

ha

ng

e)

*

R e m la r s e n

N=6 per group

Pg. 14

Remlarsen Demonstrates Target Engagement in RPE Cells in vitro

▪ Remlarsen represses collagen expression in the presence and absence of TGF-b1 in multiple human retinal pigment epithelial cell lines

UT

C

MO

CK

0.5

nM

1 n

M

5 n

M

10

nM

25

nM

MO

CK

0.5

nM

1 n

M

5 n

M

10

nM

25

nM

0

1

2

3

4

Fo

ld C

ha

ng

e

+ 5 n g /m L T G F -b 1

CO

L1

A1

CO

L1

A2

CO

L3

A1

** ****

iPS RPE Cells in Culture

N=3 per group

Pg. 15

▪ Rat STZ diabetic retinopathy model

▪ miR-29b PD biomarkers are repressed following 3x weekly IVT remlarsen treatments

▪ Rabbit proliferative vitreoretinopathy model

▪ miR-29b PD biomarkers are repressed following 4x weekly IVT remlarsen treatments

Remlarsen Demonstrates Target Engagement in the Retina in vivoin Multiple Models

S a lin e R e m la rs e n

0 .0

0 .5

1 .0

1 .5

2 .0

mR

NA

Ex

pre

ss

ion

(Fo

ld C

ha

ng

e)

* * *

COL1A1

COL1A2

COL3A1

COL4A3

CTGF

FN1

MMP2

TGFb3

B S S R e m la r s e n

0

1

2

3

mR

NA

ex

pre

ss

ion

(fo

ld c

ha

ng

e)

*

COL1A1

COL1A2

MMP2

TGFB2

N=7 per groupN=6 per group

Pg. 16

▪ miR-29b mimics are well taken up by a damaged/ulcerated cornea following topical drop administration, and by all retinal layers following intravitreal injection

▪ Remlarsen exhibits favorable PK in the cornea and retina

▪ Cornea▪ Remlarsen treatment reduces the severity of alkali burn, accelerates repair

▪ Remlarsen treatment decreases corneal hazing/scarring, corneal stromal thickness, and a-SMA staining post-burn

▪ Remlarsen engages its pharmacodynamic targets and reduces collagen/ECM expression in vivo

▪ Retina▪ Remlarsen engages its pharmacodynamic targets and reduces collagen/ECM expression in vitro

and in vivo in multiple models

▪ Remlarsen represents a potential therapeutic for corneal and retinal scarring regardless of the etiology:▪ Infectious keratitis, surgery, trauma, genetic disease

▪ AMD, DR, retinal detachment

Conclusions