Post on 22-May-2020
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