A Highly-Evolved Novel AAV Gene Therapy

Directly Addresses Fabry Disease Pathology

In Vivo by Cell Autonomous Expression in

the Heart and Other Target Organs

Abstract 140

Kevin Whittlesey1, Gabriel Brooks1, Paul Szymanski1, Ghezal Beliakoff1, Julie

Nye1, Roxanne Croze1, Chris Schmitt1, Janelle Bickta1, Katy Barglow1,

Melissa Kotterman1, Peter Francis1, David Schaffer1,2, David Kirn1

1 4D Molecular Therapeutics, Emeryville, CA

2 University of California, Berkeley, CA

Disclosures

▪ Full-time employee at 4D Molecular Therapeutics, Inc.

▪ Co-founder and owner of shares in 4D Molecular Therapeutics, Inc.

▪ Inventor on patents and/or pending patent applications related to AAV capsid variants

and AAV gene delivery.

2

Gene: GLA gene loss of function mutations lead to an

alpha-galactosidase A (AGA) enzyme deficiency.

Biologic Consequence: Insufficient AGA activity

results in accumulation of globotriaosylceramide (Gb3)

in endothelial, parenchymal and vascular smooth muscle

tissues.

Affected Organs: Heart, Kidney, Blood Vessels,

Nervous System

▪ Cardiovascular disease represents the primary

causes of death (75%)

CONFIDENTIAL3

DISEASE BIOLOGY A MULTISYSTEM DISEASE

STANDARD OF CARE

▪ Existing therapies have accelerated approval only

with no therapy obtaining full approval status

▪ Fabrazyme failed Ph4 composite-endpoint study

▪ Current therapy fails to clear Gb3 from organ

parenchymal cells or vascular smooth muscle cells

▪ ERT requires biweekly infusion

▪ Chaperone therapy benefits only 30-40% of patients

GB3 INCLUSIONS: ENDOTHELIUM

GB3: VASCULAR SMOOTH MUSCLE

PARENCHYMAL GB3 INCLUSIONSCARDIOMYOCYTE HYPERTROPHY

& VACUOLES (A), FIBROSIS (B)

A B

StrokeHearing Loss

Corneal Whorling

Pulmonary ObstructionLeft Ventricular

Hypertrophy

Kidney Failure

Acroparesthesia

Skin Rash

AbdominalpainDiarrheaNausea

Fabry Disease: An LSD with High Unmet Medical Need

Baig et al Europace. 2018 Sep 1;20(FI2):f153

Thurberg et al, Kidney Int. 2002 Dec;62(6):1933Hsu et al. Orphanet Journal of Rare Diseases 2014, 9:96

Sunder-Plassmann, G. National Kidney Foundation

Primer on Kidney Diseases (Sixth Edition),

W.B. Saunders,2014,

4D-310: Next Generation Gene Therapy Address Target

Organs in Fabry Disease

4

Endothelial Compartment Correction

GB3 INCLUSIONS: ENDOTHELIUM

GB3: VASCULAR SMOOTH MUSCLE

PARENCHYMAL GB3 INCLUSIONS

Bioreactor

Approaches

Target

Enzyme

Replacement

Anti-AGA Antibodies:

Presence are

Associated with Poor

Prognosis

Persistent Clinical Need

• Cardiomyopathy

• Renal Dysfunction

• Cerebrovascular

Disease

• Peripheral Neuropathy

• Gastrointestinal

Complaints

Whole Organ Correction

• Circumvents Challenges of Poor Uptake

of Circulating AGA

• Potentially Avoids Circulating Anti-AGA

Antibodies

• May More Fully Address Fabry Disease

Pathophysiology in Classic/Non Classic

as well as Female Fabry Disease Patients

Strategy Limitations

Thurberg et al, Kidney Int. 2002 Dec;62(6):1933

Disease-First Approach to Vector Discovery:

Therapeutic Vector Evolution

5

GENERATE MASSIVE AAV VECTOR

DIVERSITY (NUMEROUS METHODS)LEAD

CANDIDATES

DISCOVERY:

TARGET VECTOR PROFILE

IP filed:>300 capsid

variants

Tissue target, route, dose

Descending dose screening

Pre-existing antibody screening

Primate screening

15 discovery programs completed

Human Cell &

Disease ModelsIn Vivo

(NHP)

Tg1

IND

Filing

Lead Vector

CHARACTERIZATION & DEVELOPMENT

Clinical

Candidate

1st-gen wild-

type capsids, ancestral

libraries, etc.

1 BILLION unique vector

sequences in 37 libraries

4D-C102: Targets Cardiomyocytes in NHPs

6

IV 1x1013 vg/kg after 8 weeks

Primate Heart (N=30)Left atrium (3)Right atrium (3)Left ventricle (9)Right ventricle (6)Septum (9)

100% (+) GENOME DELIVERY

97% (+) PROTEIN EXPRESSION (N=30)

ROBUST CARDIOMYOCYTE PROTEIN

EXPRESSION WITH 4D-C102

IMPROVED DELIVERY VS AAV8 & AAV9

Genomes per ug DNA in heart: 4D-C102 vs AAV8 13X

Heart:Liver genome ratio 4D-C102 vs AAV8 17X

Heart:Liver genome ratio 4D-C102 vs AAV9 9X

4D-C102 Heart:Liver protein ratio 420X

Negative Control

Left Ventricle

Left Ventricle

0%

20%

40%

60%

80%

100%

NHP 1 NHP 2 NHP 3 NHP 4 NHP 5 NHP 6

AAV9 4D-C102

No Sig Tox 4D-C102

no inflammation

H&E 4x 40x

no inflammation

4D-C102

% Tissue

Sections with

Inflammation

Score >1 (4-

Point Scale)

Inflammation &

cell death

H&E 4x

AAV9

40x

Inflammation &

cell death

No Inflammation Seen with 4D-C102

Identical transgene payloads, manufacturing, dose, route

See poster #140

4D-C102: Targets Key Fabry Tissues

7 CONFIDENTIAL

0

20

40

60

80

100

20 100 500 2500

%E

GF

P+

/cT

nT

+ C

ells

MOI

***

***

***

***

AAV1

AAV8AAV9

4D-C102

* p< 0.05

4D-C102: SIG. IMPROVED HUMAN

CARDIOMYOCYTE TRANSDUCTION

4D-C102

cTnT/EGFP/DAPIPi Time: 6 days

20

100

500

2500

MOIAAV1 AAV9AAV8

not

tested

-

10,000

20,000

30,000

40,000

50,000

60,000

70,000

80,000

90,000

Right atrium Right ventricle Left atrium Left ventricle

Heart

AAV8 C102

-

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

Kidney

Kidney

AAV8 C102

-

500

1,000

1,500

2,000

2,500

3,000

3,500

4,000

4,500

Sciatic

Peripheral Nerve

AAV8 C102

gen

omes

/ug

DN

Age

nom

es/u

g D

NA

0.0E+00

5.0E+05

1.0E+06

1.5E+06

2.0E+06

2.5E+06

3.0E+06

3.5E+06

4.0E+06

Liver

Liver

AAV8 C102

See poster #140

4D-310: Target Tissue Directed Gene Therapy for Fabry

Disease

CONFIDENTIAL8

4D-C102: Robust

delivery to and

transduction of

heart, kidney, and

liver

Ubiquitous promoter

(CAG): Allows expression from

multiple cell types

GLA transgene: Codon optimization

pA

CardiomyopathyGene replacement

directly in

Cardiomyocyte to

reverse Gb3

accumulation

NephropathyGene replacement in

kidney parenchymal

cells

Liver

TransductionRobust circulating

AGA treats

endothelial cells

systemically

Capsid

Payload

C o d o n o p timize d GLA

4D-310 Increases AGA Expression & Activity

9

HUMAN FABRY PATIENT IPSC-DERIVED VENTRICULAR CARDIOMYOCYTES

NT 25 100 250

cTn

TA

GA

***p<0.004, **p=0.005 compared to NT, †† p<0.004; compared to MOI 25.

See poster #594

%cT

nT

+/A

GA

+ C

ells

100100

8080

60

40

20

0

NT 25 100 250

MOI****p<0.0001 compared to NT, ††p<0.001, †p<0.01 compared to MOI 25

4D-310: High Plasma AGA Activity

10

PLASMA AGA ACTIVITY IN 4D-310-TREATED FABRY MICE

PLASMA LYSO-Gb3 IN 4D-310-

TREATED FABRY MICE (WK 8)

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1 15 29 56

AG

A a

ctiv

ity

(nm

ol/

hr/

ml)

Days

Vehicle - WT Vehicle - KO 1E12 vg/kg

1E13 vg/kg 5E13 vg/kg

0

100

200

300

400

500

600

WT- Vehicle KO - Vehicle 1e12 vg/kg 1e13 vg/kg 5e13 vg/kg

Conc

(nM

)

*

* P<0.01 compared with vehicle-treated Fabry Mice

*

*

*

4D-310: High AGA Activity in Target Organs

11

WIDESPREAD AGA EXPRESSION IN FABRY MOUSE HEART & KIDNEYS WITH IV 4D-310

0

Vehicle 4D-310 IV

Heart

Kid

ney

TISSUE Gb3 IN 4D-310-TREATED

FABRY MICE (WK 8)

0

100

200

300

400

500

WT-Vehicle KO-Vehicle 1e12 vg/kg 1e13 vg/kg 5e13 vg/kg

Res

ponse

Rat

io

Heart Kidney Liver

4D-310: Mouse GLP Tox Study – No Significant Tox

GLP Tox Study Results

▪No sig toxicities observed

▪3 month in-life

▪132 mice treated w/ 4D-310

(up to 1.5x1014 vg/kg)

NO SIG TOXICITY WITH 4D-310 IN NORMAL MICE IN VIVO (GLP STUDY)

4D-310: Next Generation Gene Therapy For Fabry Disease

▪ 4D-310 represents a novel approach to the treatment of Lysosomal Storage Disease

▪ Therapeutic Vector Evolution was used to generate a tissue targeted capsid 4D-C102 that demonstrates:

o tropism to key target tissues in Fabry (including heart, kidney and liver)

o minimal/no inflammation

o highly efficient transduction at low doses

▪ 4D-310 (derived from C102) drives high AGA activity in the heart and kidney as well as in the systemic circulation

▪ 4D-310 allows for whole organ correction in addition to treatment of the endothelial compartment

▪ Cell autonomous AGA expression is expected to circumvent reduced activity due to Anti-AGA antibody binding

▪ No evidence of toxicity in preclinical studies: 5 Mouse studies, including GLP Tox n=132

▪ 4DMT is poised to start a clinical trial for Fabry disease with 4D-310 in 2020

13

Target Tissue Directed Gene Therapy Approach

Acknowledgments

▪ 4DMT Discovery & Engineering

▪ 4DMT HCDM

▪ 4DMT CMC

▪ 4DMT Project Management

▪ Christiane Auray-Blais, Université de Sherbrooke

CONFIDENTIAL14