18
3rd Paris NASH Symposium July 6-7, 2017
Dean Falb, Ph.D.
Co-Founder and Chief Technical Officer
Powering the Microbiome with synthetic biotics to correct metabolic dysregulation
throughout the body
3
Synthetic Biotics: A New Class of Engineered Medicines
that Operate from Our Natural Microbiome
Synthetic Biology + Bacteria = Synthetic Biotic
Therapeutic delivered
locally to treat systemic
diseases
Synthetic
• Engineered bacteria
• With designed genetic circuits
• To degrade metabolites that induce
disease or synthesize substances to
treat disease
Biotics: E. coli Nissle as chassis:
• Widely-used oral probiotic
• Leverage the safety of probiotic
• Found within natural human
microbiome
• Amenable to genetic manipulation
4
Probiotic bacteria: E. coli Nissle
SYNB1020: Conversion of Toxic Ammonia into Beneficial
Arginine for the Treatment of UCD and HE
Synthetic Biotic
Correct the deficiency
✓
Arginine argE argC argB argH
R FNR FNR carA carB argG argl argD fbr-argA
Ammonia
Metabolic Conversions
Arginine
Synthetic Genetic Circuit
Glutamate
UCD and HE Patients
Target Profile:
Normalize
Ammonia Levels
Dietary Protein
Liberalization
5
SYNB1020: Efficient Ammonia Conversion by Synthetic Biotic In
Vitro and In Vivo
Blood Ammonia Reduced in vivo (spf-ash hyperammonia/UCD model
on High Protein Diet)
0
100
50
150
200
250
Blo
od
Am
mo
nia
(u
M) ***
NC – Normal chow, HP – High protein chow
SYNB1010 – arg producing, Thy A auxotrophy, Kan resistant (a
kanamycin resistant version of SYNB1020 clinical candidate)
Ammonia to Arginine Conversion
in vitro
Nissle – E. coli Nissle 1917 Strep resistant control strain
SYNB1010 –arginine producing, Thy A auxotrophy, Kan resistant
SYNB1020, arginine producing, Thy A auxotrophy, clinical
candidate
Mean Arg production
rate=650nmoles/109 cells/hr
Arg
inin
e P
rod
uc
tio
n (
nm
ole
s/1
09c
ells)
Time (min)
20 40 60 80 100 120
250
500
750
1000
1250
1500
Nissle
SYNB1010
SYNB1020
6
SYNB1618: Degradation of Toxic Phenylalanine
for the Treatment of PKU
Probiotic bacteria: E. coli Nissle
Synthetic Biotic
Correct the deficiency
✓
FNR FNR PAL3
Phenylalanine t-Cinnamic Acid
Synthetic Genetic Circuit
Phenylalanine t-Cinnamic Acid
FNR FNR pheP Metabolic Conversions
High-
Affinity
Uptake
AraC AraC FNR LAAD
Phenylketonuria Patients
Hippurate
7
SYNB1618: Efficient Phe Degradation In Vitro and In Vivo
Dose Response of SYNB1618 in PKU Mice
Each bar represents an average of n=9 mice/group
Ph
en
yla
lan
ine m
eta
bo
lized
(to
tal
um
ol)
/mo
use
8
Metabolic Disease and the Gut Microbiome
• FMT study in obese patients with
Metabolic Syndrome
• Placebo-controlled study in 18 male
patients
• Treatment group (n=9) received
allogeneic microbiota from lean donors
• Placebo group (n=9) received
autologous microbiota
9
Synthetic Biotic Approaches to Treating NASH
NASH Pathology: • Inflammation
• Fibrosis
• Insulin resistance
• Hyperlipidemia
• Obesity
• SCFA Production
- Improves barrier function
- Induces Treg differentiation
- Lowers inflammation
• GLP-1 Secretion - Improves insulin secretion
- Promotes weight loss
- Improves lipid profiles
• Bile salt modulation - Lower lipid absorption
- Lowers bile acid concentrations
10
Butyrate Production in E. coli Nissle
CONFIDENTIAL
NAD+
FNR 1.18.1.2
PntB 1.6.1.2
NADH
Fdred2- Fdsx
NADP+ NADPH
Butyrate
Butyryl-P
Butyryl-CoA Crotonyl-CoA
S-3-hydroxybutyryl-CoA
Acetoacetyl-CoA
Krebs Cycle
Acetate
Gly
coly
sis
Glucose
NADH
CoA-SH
Phosphate
H2O
NAD+
NADH
CoA-SH
CRT2 (4.2.1.17)
HBD (1.1.1.157)
BCD2/ETF (1.3.99.2)
PBT (2.3.1.19)
BUK (2.7.2.7) ADP
ATP
THIA1 (2.3.1.9)
NAD+
Acetyl-CoA
pLOGIC046 –
ter/tesB
5700 bp
RBS and leader region
M13 fwd
RBS
1000
2000
3000
4000
5000
• Iterative pathway improvements increased
butyrate yield and reduced circuit size
• Replacement of Ptet with PfnrS retains high
level production
11
Lead Optimization of Butyrate Producing Synthetic
Biotic Strains
0
1
2
3
4
5
6
7
8
mM
/OD
(2
4h
)
0
5
10
15
20
25
+O2 -O2 -O2 -O2 -O2
NissleSYN363/RCM
CMB588 TYRO BUTYR
Buty
rate
(m
M)
2h 8h 24h 56h
CONFIDENTIAL
Iterative
engineering on
plasmid vectors
increases
butyrate yield
Conversion of
Tet to FNR
promoters with
subsequent
integration
retains strong
production
Anaerobically-induced Synlogic strain
produces butyrate at similar levels to
naturally-producing Clostridia strains
(CMB588, C. tyrobutylicum (TYRO), C.
butyricum (BUTYR)
12
H20
Contr
ol
H20
(+)1
00m
M B
utyra
te
SYN
94
SYN
363
0
1
2
3
4
[Bu
tyra
te]
in m
M
p=0.04
p=0.03
Administration of SYN363 results in significant increases
in fecal butyrate levels
13
Butyrate Effects in DSS IBD Model
H20
DS
S 0
.5%
DS
S 0
.5%
BU
TY
RA
TE
100m
M
DS
S 0
.5%
SY
N94
DS
S 0
.5%
SY
N363
0
5 0
1 0 0
1 5 0
D S S 0 0 4 L C N 2 E L IS A
LC
N2
(n
g/g
)
H20
DS
S 0
.5%
DS
S 0
.5%
BU
TY
RA
TE
100m
M
DS
S 0
.5%
SY
N94
DS
S 0
.5%
SY
N363
0
1 0
2 0
3 0
D S S 0 0 4 C A L P R O T E C T IN E L IS A
Ca
lpro
tec
tin
S1
00
A8
/9 (
ng
/g)
H20 DSS DSS/
Buytr
DSS/
SYN94
DSS/
SYN363
• Calprotectin and LCN2 measured
in fecal samples
• Produced by activated neutrophils
in the mucosa
• Surrogate for gut permeability
• Used for clinical assessment of
IBD
Groups - H2O control
- DSS alone
- DSS + oral butyrate
- DSS + SYN94 (native probiotic)
- DSS + SYN363 (butyrate strain)
14
Modulating GLP1 secretion levels with RBS
SYN2627 SYN26430
10
20
30
40
50
GL
P-1
(n
g/m
l)
30C
Supernatant
Cellular
PhoASS GLP1
SYN2627
∆lpp
TetR PhoASS GLP1
SYN2643
∆lpp
TetR
15
Bile Salt Hydrolases: Consuming Primary Bile Salts
Inducible bile salt hydrolase operon
Begley M et al. Appl Environ Microbiol. (2006)
Primary Bile Acid
BSH Activity
tetR BSH
2
aTc
Ptet
BSH
1
Joyce, S. A., et al. (2014). PNAS.
BSH1 and BSH2 activity relative to E. coli-only
control; measured as rate of taurine release
BSH1 and BSH2 from
Lactobacillus salivarius
NASH patients have elevated blood bile
salt concentration, which may be involved
with liver damage.
16
0 1 2 240
1
2
3
Time [h]
arg
inin
e m
M
Arginine production
SYN363
SYN955
SYN1536
SYN363 – E. coli Nissle harboring
butyrate plasmid (Pfnr-ter-pbt-buk)
SYN955 - E. coli Nissle dArgR;
malEK:PfnrS-fbrArgA; thyA::frt
SYN1536 – SYN955 harboring
butyrate plasmid (Pfnr-ter-pbt-buk)
0 1 2 24
0
5
10
15
Time [h]
Bu
tyra
te m
M
Butyrate production
SYN363
SYN955
SYN1536
Simultaneous and efficient performance of an Integrated
NH4+/Butyrate Dual Synthetic Biotic
1Mb
2M
b 3Mb
4Mb
5Mb 5.4Mb
Nissle
Chromosome
ΔthyA
FNR-fbr-argA ::malE/K
ΔargR
FNR-butyrate::agaI/rsmI
17
Decrease disease burden
Synthetic Biotic for NASH/PBC
Synthetic Biotic
Synthetic Genetic Circuit
Probiotic bacteria: E. coli Nissle
✓
NASH pathogenesis may benefit
from a synthetic biotic with
multiple mechanisms of action,
including up-regulating butyrate,
propionate, and GLP-1 production
Armstrong, Matthew J., et al. "Glucagon-like
peptide 1 decreases lipotoxicity in non-alcoholic
steatohepatitis." Journal of hepatology (2015).
Chambers, Edward S., et al. "Effects of targeted
delivery of propionate to the human colon on
appetite regulation, body weight maintenance
and adiposity in overweight adults." Gut (2014):
gutjnl-2014.
Jin, Cheng Jun, et al. "Supplementation of
sodium butyrate protects mice from the
development of non-alcoholic steatohepatitis
(NASH)." British Journal of Nutrition 114.11
(2015): 1745-1755.
Butyrate
FNR FNR Butyrate
FNR FNR BSH
FNR FNR GLP-1
GLP-1
Bile Salt Hydrolase
NASH Patients
