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Development of an AAV-Based Gene

Therapy for Children With

Congenital Hearing Loss Due to

Otoferlin Deficiency (DB-OTO)

Adam Palermo

ASGCT

May 11, 2021

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This presentation contains forward looking statements that involve substantial risks and uncertainties. All statements, otherthan statements of historical facts, contained in this presentation, including statements regarding the Company’s strategy, future operations, future financial position, future revenues, projected costs, prospects, plans and objectives of management, are forward looking statements. The words “anticipate,” “believe,” “continue,” “could,” “estimate,” “expect,” “intend,” “may,” “might,” “plan,” “potential,” “predict,” “project,” “should,” or “would,” or the negative of these terms, or other comparableterminology are intended to identify forward looking statements, although not all forward looking statements contain these identifying words. The Company may not actually achieve the plans, intentions or expectations disclosed in these forward-looking statements, and you should not place undue reliance on these forward-looking statements. Actual results or events could differ materially from the plans, intentions and expectations disclosed in these forward-looking statements. In addition, the forward-looking statements included in this presentation represent the Company’s views as of the date of this presentation. The Company anticipates that subsequent events and developments will cause its views to change. However, while the Company may elect to update these forward-looking statements at some point in the future, it specifically disclaims any obligation to do so. These forward-looking statements should not be relied upon as representing the Company’s views as of any date subsequent to the date of this presentation.

Forward-Looking Statements

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Background

on Otoferlin

Deficiency

Stimulus

Inner hair

cell

Neuron

Signal

transmission

Synaptic ribbon Synaptic ribbon

Synaptic

vesicles

Synapse OtoferlinReleased

Neurotransmitter

Otoferlin

tethered to

membrane

Ca2+

Otoferlin deficiency secondary to biallelic mutations of the OTOF gene causes permanent congenital severe-to-profound deafness. Otoferlin is expressed in the primary sensory receptors of the ear (the inner hair cells) and enables synaptic transmission between the inner hair cells and the dendrites of the auditory nerve by functioning as a calcium sensor for exocytosis during synaptic vesicle release, vesicle trafficking, and replenishment. Of the 1 in 500 neonates who are born with hearing loss annually in the US, 50 to 200 are caused by Otoferlin deficiency.

There are no approved therapies for Otoferlin deficiency; infants with biallelic OTOF mutations are currently managed with assistive devices.

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DB-OTO: Dual AAV to Rescue Otoferlin Deficiency

5’ OTOF-cDNA

3’ OTOF-cDNA

IHC

5’ OTOF-cDNA 3’ OTOF-cDNA

Full-length OTOF vector gene

OTOF mRNA

Otoferlin protein

We have developed an Adeno-Associated Virus (AAV)-based gene transfer therapy for rescue of hearing in an Otoferlin deficiency model that mimics a common human OTOF mutation (OTOFQ828X/Q828X). Because human OTOF cDNA exceeds the packaging capacity of a standard AAV, we used a dual AAV system to locally deliver Otoferlin to the inner ears of OTOFQ828X/Q828X mice.

Key considerations for vector development included the human OTOF isoform that should be expressed, the promoter sequence that determines in which cells the human OTOF will and will not be expressed, the recombinogenic region that enables the dual AAV system, and the capsid in which these elements are packaged for efficient delivery into the target cells.

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Novel OTOFQ828X/Q828X Disease Model Recapitulates Human

Phenotype

Homozygotes

Heterozygotes

• Q829X is a common and well-characterized pathogenic

mutation in Spanish and Latin American populations.

• We generated a mouse disease model by generating

the orthologous mouse mutation, Q828X.

• Hearing thresholds, as measured by auditory brainstem

response (ABR; upper right), were elevated.

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DB-OTO Design ConsiderationsDual AAV1 Vectors with Cell-Selective Myo15 Promoter Controlling Expression of hOTOF

CONFIDENTIAL

Serotype Coding Sequence

Promoter 5’ CDS AP

3’ CDS pAAP

Promoter 5’ CDS AP

3’ CDS pAAP

AAV1 demonstrates broad tropism and

high infectivity in the NHP cochlea.

Outer Hair Cell

Auditory

Nerve

Supporting

Cell Inner

Hair Cell

Outer Hair Cell

Supporting

Cell Inner

Hair Cell

Auditory

Nerve

promoter Transgene

• Goal = restrict transgene expression to target

cell types only (particularly if ectopic expression

of transgene is deleterious outside target cells)

• Design informed by bioinformatics

• Promoter strength may be important to fine tune

transgene levels in transduced cells

Scale

chr11:

TSS peaks

RepeatMasker

5 kb mm10

60,470,000 60,471,000 60,472,000 60,473,000 60,474,000 60,475,000 60,476,000 60,477,000 60,478,000 60,479,000

mMyo15_intron_minimal

Myo15

Myo15

Myo15

Total counts100 -

0 _

Max counts100 -

0 _

Atoh1+InnerEarHairCellsOrganOfCortiPl1+

Sox2+SupportingCellsOrganOfCortiPl1+

uHC

25 -

0 _

uSC

25 -

0 _

Placental Cons

Scale

chr11:

TSS peaks

RepeatMasker

5 kb mm10

60,470,000 60,471,000 60,472,000 60,473,000 60,474,000 60,475,000 60,476,000 60,477,000 60,478,000 60,479,000

mMyo15_intron_minimal

Myo15

Myo15

Myo15

Total counts100 -

0 _

Max counts100 -

0 _

Atoh1+InnerEarHairCellsOrganOfCortiPl1+

Sox2+SupportingCellsOrganOfCortiPl1+

uHC

25 -

0 _

uSC

25 -

0 _

Placental Cons

Scale

chr11:

TSS peaks

RepeatMasker

5 kb mm10

60,470,000 60,471,000 60,472,000 60,473,000 60,474,000 60,475,000 60,476,000 60,477,000 60,478,000 60,479,000

mMyo15_intron_minimal

Myo15

Myo15

Myo15

Total counts100 -

0 _

Max counts100 -

0 _

Atoh1+InnerEarHairCellsOrganOfCortiPl1+

Sox2+SupportingCellsOrganOfCortiPl1+

uHC

25 -

0 _

uSC

25 -

0 _

Placental Cons

ATAC-Seq

Conservation Mouse explant

AAV1 Myo15 GFP 1E11 vg/culture

Proprietary promoter restricts

expression of GFP reporter to hair cells.

Myo7a hair cells

GFP reporter

Mouse explant

AAV1 UbC GFP 1E11 vg/culture

Non-human primate cochlea

AAV1 Myo15 GFP 1E12 vg/ear

V1 isoformDose: 3E9 vg/ear

AAV2quad V1-CO AAV2quad V5-CO0

20

40

60

80

100

120

AB

R T

hre

sh

old

at

22

.6 k

Hz (

dB

SP

L)

No Response at Equipment Limits

Range of normal hearing

V5 isoformDose: 7E9 vg/ear

Human OTOFv5, but not OTOFv1,

rescues hearing in OTOFQ828X/Q828X mice.

V1V4V3V2V5

OTOF splice variant V5 is the predominant

isoform in the NHP and mouse cochlea.

Promoter

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DB-OTO Delivery Rescues Normal Hearing Sensitivity in

Congenitally Deaf Mice

DB-OTO rescues otoferlin protein expression in Q828X

mouse inner hair cells.

DB-OTO rescues auditory brainstem response in

Q828X mouse.

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DB-OTO-Driven Recovery is Durable when Dosed at 4 Weeks of

Age

0 10 20 30 400

20

40

60

80

100

120

mean ABR threshold at 22.6 kHz

Time after treatment (wks)

AB

R T

hre

sh

old

(d

B S

PL

) no neural response at equipment limit

Otof-Q828X wt

Otof-Q828X het

Otof-Q828X mut

Treated Otof-Q828X muthom

hom

OtofQ828X/Q828X = hom; OtofQ828X/+ = het; and Otof+/+ = wt

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DB-OTO Also Rescues Hearing in Mice when Dosed at 32 and 52

Weeks of Age

8 11.3 16 22.6 32 Click0

10

20

30

40

50

60

70

80

90

100

110

4W ABR Threshold (only good DPOAE)

Tone Frequency (kHz)

Th

resh

old

(d

B)

OTOF-10039 52w old 4W vehicle (n=5)

OTOF-10039 32w old 4W vehicle (n=5)

OTOF-10039 1kB-Myo15-hOtof 32W old 4W(n=6)

OTOF-10039 1kB-Myo15-hOtof 52W old 4W(n=6)

Vehicle, 32 wk

Vehicle, 52 wk

DB-OTO, 32 wk

DB-OTO, 52 wk

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OTOF Expression in >20% Inner Hair Cells Restores Normal ABR

Sensitivity in Q828X Mice

No improvement Partial improvement Within normal limits

Fra

ctio

n of

anim

als

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Translation to Non-Human Primates

AAV1-Myo15-dual GFPAAV1-Myo15-dual hOTOF

FFPE sections; ISH & IHCWholemount; immunofluorescence

In order to show that efficacious DB-OTO mouse doses are safely translatable to non-human primates, we dosed the right ears of 6 primates with DB-OTO. We dosed the contralateral ears with a dual AAV construct equivalent to DB-OTO, but with the otoferlin coding sequence replaced with GFP.

In this study, we looked at impact on hearing, expression of GFP quantitatively and human otoferlin qualitatively, and biodistribution outside the ear.

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DB-OTO has Minimal Impact on Wild Type Primate Hearing

Thresholds

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AAV1-Myo15-dual GFP drives robust expression in inner hair cells in

injected NHPs

2 kHz

8 kHz

Vehicle

control

(no AAV)

16 kHz

32 kHz

AA

V1-

Myo

15-d

ualG

FP

Primate vocalization

frequencies

4 kHz

1 kHz

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Outside of the cochlea, AAV genomes were detected in spleen and lymph

node, but not appreciably in other organs

ddPCR- based detection of vector genomes

eGFP DNAScope

1103 1104 1105 1006 3101

0

50

100

150

200

Spleen 5' vector genomes

Animal ID

Co

pie

s M

yo

15/n

g in

pu

t D

NA

1103 1104 1105 1006 3101

0

50

100

150

200

Lumbar DRG, 5' vector genomes

Animal ID

Co

pie

s M

yo

15/n

g in

pu

t D

NA

1101 Cervical Lymph Node closeup 1102 Spleen closeup

L994 brain closeupbrainSpinal cord Spinal cord

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Little to No Impact of Pre-Existing anti-AAV Capsid NAbs on

Dual AAV Expression in the NHP Inner Ear

0

50

100

256 512 1024 2048 4096

GFP versus serum

Serum NAb levels (IC50)

%G

FP

+ c

ells, 1-1

1.3

kH

z

No c

all

Serum NAb Titers vs Transduction

Anim

al =

"10

06"

Anim

al =

"11

01"

Anim

al =

"11

02"

Anim

al =

"11

03"

Anim

al =

"11

04"

Anim

al =

"11

05"

Anim

al =

"31

01"

0

1000

2000

3000

4000

AAV1 Neutralizing Antibody Levels

NA

b t

iter

(IC

50)

Pre-injection

Day 14

Day 28

Upper limit of detection

SalineAAV1-treated

Serum Neutralizing Antibody Titers

Acceptable NAb levels for

systemic delivery

sero

negative

sero

negative

1 2 3 4 5 6 VehAnimal:

AAV-Treated

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Conclusions

• DB-OTO is an efficacious treatment for otoferlin-deficient hearing loss in

Q828X mice.

• In NHP, DB-OTO and the surgical administration procedure were generally

well-tolerated, and dual AAV was able to drive selective expression of GFP

in the majority of inner har cells.

• We intend to initiate a human phase 1/2 clinical trial in 2022.

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• To address some of the barriers to receiving genetic diagnosis, we have launched

Amplify™: a next generation sequencing panel that includes 203 known hearing loss-

associated genes and is performed in a CLIA certified laboratory by Invitae

• Clinicians who identify a child with auditory neuropathy can offer the parents the

opportunity to receive genetic testing for the child on the Amplify™ comprehensive HL

gene panel at no cost