Powering the Microbiome

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Theodore Parks
6 years ago
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2 Powering the Microbiome with synthetic biotics to correct metabolic dysregulation throughout the body 3 rd Paris NASH Symposium July 6-7, 217 Dean Falb, Ph.D. Co-Founder and Chief Technical Officer

Synthetic Biotics: A New Class of Engineered

Synthetic Engineered bacteria With designed genetic

or synthesize substances to treat disease Biotics:

probiotic Leverage the safety of probiotic Found

manipulation Synthetic Biology + Bacteria =

3 Synthetic Biotics: A New Class of Engineered Medicines that Operate from Our Natural Microbiome 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 Synthetic Biology + Bacteria = Synthetic Biotic Therapeutic delivered locally to treat systemic diseases 3

4 SYNB12: Conversion of Toxic Ammonia into Beneficial Arginine for the Treatment of UCD and HE Ammonia Glutamate Arginine UCD and HE Patients arge argc argb argh Metabolic Conversions Arginine R FNR FNR fbr-arga argd argl argg Synthetic Genetic Circuit cara carb Synthetic Biotic Correct the deficiency Probiotic bacteria: E. coli Nissle Target Profile: Normalize Ammonia Levels Dietary Protein Liberalization 4

5 Arginine Production (nmoles/1 9 cells) Blood Ammonia (um) SYNB12: Efficient Ammonia Conversion by Synthetic Biotic In Vitro and In Vivo Ammonia to Arginine Conversion in vitro Blood Ammonia Reduced in vivo (spf-ash hyperammonia/ucd model on High Protein Diet) Nissle 2 *** SYNB11 1 SYNB Mean Arg production rate=65nmoles/1 9 cells/hr Time (min) Nissle E. coli Nissle 1917 Strep resistant control strain SYNB11 arginine producing, Thy A auxotrophy, Kan resistant SYNB12, arginine producing, Thy A auxotrophy, clinical candidate NC Normal chow, HP High protein chow SYNB11 arg producing, Thy A auxotrophy, Kan resistant (a kanamycin resistant version of SYNB12 clinical candidate) 5

SYNB1618: Degradation of Toxic Phenylalanine for the Treatment of PKU Phenylalanine High- Affinity Uptake Phenylalanine FNR FNR phep t-cinnamic Acid FNR FNR PAL3 Metabolic

6 SYNB1618: Degradation of Toxic Phenylalanine for the Treatment of PKU Phenylalanine High- Affinity Uptake Phenylalanine FNR FNR phep t-cinnamic Acid FNR FNR PAL3 Metabolic Conversions Hippurate t-cinnamic Acid Synthetic Biotic Phenylketonuria Patients AraC AraC FNR LAAD Synthetic Genetic Circuit Correct the deficiency Probiotic bacteria: E. coli Nissle 6

7 Phenylalanine metabolized (total umol)/mouse 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 7

male patients Treatment group (n=9) received allogeneic

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 8

Synthetic Biotic Approaches to Treating NASH NASH Pathology: Inflammation Fibrosis Insulin resistance Hyperlipidemia Obesity SCFA Production - Improves barrier function - Induces Treg

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 9

10 Glycolysis Butyrate Production in E. coli Nissle Acetate Glucose Acetyl-CoA Krebs Cycle Iterative pathway improvements increased butyrate yield and reduced circuit size Replacement of P tet with P fnrs retains high level production Butyrate ATP ADP Butyryl-P CoA-SH Phosphate BUK ( ) Butyryl-CoA NAD + PBT ( ) NAD + Fd red 2- THIA1 CoA-SH ( ) Acetoacetyl-CoA NADH BCD2/ETF ( ) FNR Fd sx S-3-hydroxybutyryl-CoA H 2 O Crotonyl-CoA NADH HBD ( ) CRT2 ( ) NADH NAD M13 fwd plogic46 ter/tesb 57 bp RBS RBS and leader region 2 1 NADP + PntB NADPH 3 1 CONFIDENTIAL

11 Lead Optimization of Butyrate Producing Synthetic Biotic Strains mm/od (24h) Butyrate (mm) Iterative engineering on plasmid vectors increases butyrate yield Conversion of Tet to FNR promoters with subsequent integration retains strong production +O2 -O2 -O2 -O2 -O2 Nissle SYN363/RCM 2h 8h 24h 56h CMB588 TYRO BUTYR Anaerobically-induced Synlogic strain produces butyrate at similar levels to naturally-producing Clostridia strains (CMB588, C. tyrobutylicum (TYRO), C. butyricum (BUTYR) 11 CONFIDENTIAL

12 Administration of SYN363 results in significant increases in fecal butyrate levels [Butyrate] in mm 4 p=.4 p= H2 Control H2(+)1mM Butyrate SYN94 SYN363 12

13 C a lp r o te c tin S 1 A 8 /9 (n g /g ) L C N 2 (n g /g ) Butyrate Effects in DSS IBD Model D S S 4 L C N 2 E L IS A 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) D S S 4 C A L P R O T E C T IN E L IS A H 2 D S S.5 % D S S.5 % B U T Y R A T E 1 m M H 2 D S S.5 % R A T E 1 m M D S S.5 % S Y N 9 4 H2 DSS DSS/ Buytr S.5 % S Y N 9 4 D S S.5 % S Y N DSS/ SYN94 DSS/ SYN363.5 % S Y N 3 6 3

14 GLP-1 (ng/ml) Modulating GLP1 secretion levels with RBS SYN2627 SYN2643 TetR PhoA SS GLP1 TetR PhoA SS GLP1 lpp lpp 3C Cellular Supernatant SYN2627 SYN

15 Bile Salt Hydrolases: Consuming Primary Bile Salts NASH patients have elevated blood bile salt concentration, which may be involved with liver damage. Inducible bile salt hydrolase operon atc Primary Bile Acid tetr BSH Ptet 1 BSH 2 BSH1 and BSH2 from Lactobacillus salivarius BSH1 and BSH2 activity relative to E. coli-only control; measured as rate of taurine release BSH Activity 15 Joyce, S. A., et al. (214). PNAS. Begley M et al. Appl Environ Microbiol. (26)

16 Simultaneous and efficient performance of an Integrated NH 4+ /Butyrate Dual Synthetic Biotic arginine mm Butyrate mm Arginine production Butyrate production SYN363 SYN955 SYN SYN363 SYN955 SYN Time [h] Time [h] 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) FNR-fbr-argA ::male/k 4Mb ΔargR FNR-butyrate::agaI/rsmI ΔthyA 3Mb 5Mb 5.4Mb Nissle Chromosome 2M b 1Mb 16

17 Synthetic Biotic for NASH/PBC Butyrate FNR FNR Butyrate FNR FNR BSH Bile Salt Hydrolase GLP-1 NASH Patients FNR FNR GLP-1 Synthetic Genetic Circuit Synthetic Biotic Decrease disease burden 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 (215). 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 (214): gutjnl-214. Jin, Cheng Jun, et al. "Supplementation of sodium butyrate protects mice from the development of non-alcoholic steatohepatitis (NASH)." British Journal of Nutrition (215):

18 Questions? Synlogictx.com 18 CONFIDENTIAL

A Novel Class of Living Medicines

A Novel Class of Living Medicines Synthetic Biotic TM medicines to perform and deliver critical therapeutic functions to treat diseases throughout the body Genetically Engineered E. coli Nissle attenuates