Therapeutic Programming of Synthetic Biotic Medicines

Transcription

1 Therapeutic Programming of Synthetic Biotic Medicines Synthetic Biotic TM medicines to perform and deliver critical therapeutic functions to treat diseases throughout the body An Engineered E. coli Nissle for the treatment of Phenylketonuria (PKU) Vincent Isabella Senior Scientist June 11 th, 2018

2 Synthetic Biotic Medicines: A Novel Class of Living Medicines Synthetic Engineered bacteria With designed genetic circuits To degrade metabolites that induce disease or synthesize substances to treat disease Biotic: 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 Medicine Therapeutic delivered locally to treat systemic diseases 2

3 Synlogic Synthetic Biotic Platform: Bringing Rational Drug Development to the Microbiome Build Potency Rational design: Synthetic biology tools applied Engineer potency Exceed endogenous bacterial activity Apply Pharmacological Principles Pharmacologically tractable: Non-colonizing Measurable doseresponse Develop Reliable Manufacturing GMP manufacturing: Single strain Reproducible yield Formulation & delivery 3

4 SYNB1618 for Phenylketonuria (PKU): Facilitating Normalization of Plasma Phe Levels Rare Inherited amino acid metabolism disorder Build up of amino acid phenylalanine (Phe) in the blood and organs caused by mutation/loss of function of Phenylalanine hydroxylase (PAH), which normally converts Phe to Tyr Diagnosed: 16,500 in US, similar in Europe If left untreated, symptoms include cognitive impairment, convulsions, behavior problems, skin rash, musty body odor Treatment: Low protein diet (no meat, dairy, nuts, eggs) Difficult to maintain lifelong compliance Kuvan: PAH cofactor (Only for patients with some residual PAH activity) Cofactor of PAH enzyme (20-40% of patients are responsive) 4

5 SYNB1618 Mechanism of Action: Designed to Convert Toxic Phenylalanine to non-toxic metabolites Phenylalanine PheP trans-cinnamate High Affinity Uptake trans-cinnamate FNR FNR phep Phenylalanine PAL FNR FNR PAL PAL dapa P tac Key strain design elements: PAL (Phenylalanine ammonia lyase) Breaks down Phe to non toxic byproduct, trans-cinnamate (TCA) phep High affinity Phe transporter increase rate of Phe uptake into engineered cells, alleviating transport bottleneck FNR (fumarate and nitrate reductase regulator) promoter Activates transcription of payload in vivo ΔdapA auxotrophy as biocontainment element 5

6 SYNB1618 Mechanism of Action: Designed to Convert Toxic Phenylalanine to Trans-cinnamic Acid Healthy Phe Phenylalanine Hydroxylase (PAH): converts Phe into Tyrosine Tyrosine Amino acids from protein [Absorption and Recirculation] PKU! SYNB1618 Impaired PAH Accumulation of Phe to toxic levels Phenylalanine High- Affinity Uptake Phenylalanine FNR FNR phep FNR FNR PAL3 Synthetic Genetic Circuit t-cinnamic Acid Metabolic Conversions Probiotic bacteria: E. Coli Nissle Normalized levels of Phe When Phe is not efficiently metabolized (PKU) SYNB1618 provides an alternative mechanism 6

7 Mechanism of Action: Functional analysis of PAL and phep in vitro in E. coli Nissle TCA production Phe degradation Expression of PAL leads to production of trans-cinnamate (TCA) as a product of Phe degradation Uptake of Phe is rate-limiting; Expression of transporter, phep, led to a 7-fold increase in the rate of TCA production/phe degradation 7

8 Pah enu2/enu2 : A mouse model of PKU Profiling the mouse model and the small intestine as a Phe sink A Standard diet (SD) vs. Phe-deficient diet (PDD) in Pah enu2/enu2 mice B Stable elevation of Phe post-sq injection in Pah enu2/enu2 mice S e ru m P h e (m M ) S e ru m P h e (m M ) E N U 2 - S D E N U 2 - P D D T im e (h ) C High Phe in small intestine of Pah enu2/enu2 mice D High Phe in small intestine of C57BL/6 mice h 0.3 h 2 h h 0.5 h 1 h 2 h P h e (m M ) 4.0 P h e (m M ) S e ru m S m a ll In te s tin e L a r g e I n t e s t i n e S e ru m S m a ll In te s tin e C e c u m L a r g e I n t e s t i n e 8

9 Dietary vs Non-Dietary sources of Phe Enterorecirculation as a source of free Phe Circulatory system Enterorecirculation and Phe: Salivary glands Stomach Intestinal epithelium Secretions Liver Dietary protein is not the only source of Phe in small intestine Small intestine Endogenous proteins g/day Digestive enzymes Mucins Serum albumin Immunoglobulins Epithelial proteins Dietary proteins g/day Proteases Proteases Free amino acids Oligopeptides Portal blood Chang TM, Nature Reviews in Drug Discovery, 2005 Dave AL et al., Peptides, 2016 Amino acids recycled into the GI tract for reabsorption High steady-state levels of free Phe in the small intestine 9

10 Enterorecirculation of Phe in mice Isotopically-labeled Phe in blood appears in the GI tract n = 4 n = 4 n = 4 n = 4 n = 12 or C 6 -Phe (SQ) T0 T1 T2 T3 A ( 13 C 6 )-Phe in the small intestine of PKU mice B ( 13 C 6 )-Phe in the small intestine of WT mice h 0.3 h 2 h h 0.5 h 1 h 2 h C -P h e (m M ) C -P h e (m M ) S e ru m S m a ll In te s tin e L a r g e I n t e s t i n e S e ru m S m a ll In te s tin e C e c u m L a r g e I n t e s t i n e Enterorecirculation and Phe: Phe delivered to the blood was found in the small intestine of both PKU and WT mice as early as 20 min 10

11 Hippurate (HA): A biomarker of SYNB1618 activity in vivo The fate of orally dosed TCA 8 0 T C A G a v a g e d ( o r a l) H A e x c r e te d ( u r in e ) Phenylalanine trans-cinnamate Liver enzymes SYNB1618- specific T C A o r H A ( µ m o l) Hippurate Urine G r o u p 1 G r o u p 2 G r o u p 3 G r o u p 4 G r o u p 5 Essentially all orally dosed TCA recovered as urinary hippurate (HA) HA could serve as a biomarker of SYNB1618 activity in vivo 11

12 Dose-dependent activity of SYNB1618 in Pah enu2/enu2 mice: HA is a biomarker of SYNB1618 activity in vivo Urine collection over 4h for determination of HA recovery 12 Each dot represents urine collected from a metabolic cage of 3 mice/cage

13 In vivo efficacy of SYNB1618 in Pah enu2/enu2 mice SYNB1618 reduces enterorecirculating Phe with concomitant production of urinary HA A Bleed 1 0h n = 9/group; PDD Phe (SQ) B Dose 1 Dose 2 Dose 3 Bleed 2 1h 2h 3h 4h B Urine Each dot represents an individual PKU mouse Each dot represents collection from a metabolic cage of 3 mice/cage Results: Enterorecirculating (non-dietary) Phe is accessible within the GI tract; its degradation can lead to significant serum Phe reduction 13

14 In vivo activity of SYNB1618 in healthy non-human primates Evidence for a Phe sink and enterorecirculation in a primate model Remove Food Peptone (or water) SYNB1618 (or vehicle) IV 13 C 6 -Phe (Group 1) Urine -16h 0h 1h 6h B A B Results: Significant HA recovered from fasted animals, even those that did not receive a peptide bolus IV 13 C 6 -Phe was recovered in the urine as 13 C 6 -HA, demonstrating enterorecirculation and SYNB1618 activity 14

15 In vivo efficacy of SYNB1618 in healthy NHPs SYNB1618 results in significant blunting in serum Phe following challenge Remove Food d 5 -Phe (70mg) SYNB1618 (or vehicle) Urine -16h B 0h 0.5h 1h 2h Bleed Bleed Bleed Bleed Bleed Bleed 4h 6h A B d 5 -H A (µ m o l) d 5 -P he d 5 - P h e / S Y N B Results: SYNB1618 administration led to significant decrease (p = 0.015) in d 5 -Phe AUC with corresponding increase in d 5 - HA recovered in the urine 15

16 Dose-responsive activity of SYNB1618 in healthy NHPs SYNB1618 exhibits dose-dependent pharmacokinetics Results: Dose-responsive recovery of urinary HA Dose-responsive serum AUC for TCA and HA Significant blunting of serum Phe elevation at the 3 highest doses of SYNB1618 (p < 0.05) 16

17 Conclusions A chromosomally integrated, modified E. coli Nissle strain, SYNB1618, was created and could degrade Phenylalanine to the non-toxic product trans-cinnamate Activity of the strain could be enhanced by co-expression of high affinity transporter, phep Phenylalanine is abundant in the small intestine Both dietary and non-dietary Phe make up a reservoir of Phe in the GI tract Phe from the blood can re-enter the GI tract through enterorecirculation The product of SYNB1618, trans-cinnamate, is converted to hippurate and excreted in urine, which can be used as a quantitative biomarker of in vivo strain activity SYNB1618, administered orally, can result in significant decreases in serum Phe in both mice and NHP SYNB1618 also exhibits dose-responsive pharmacokinetics SYNB1618 has entered Phase 1 trials in healthy volunteers 17

18 Acknowledgements Discovery David Lubkowicz Adam Fisher Sarah Rowe Yves Millet Cami Anderson Pharmacology Binh Ha Denise Wong Bioanalytical Mary Castillo Michael James Process Devlopment Pip Reeder Munira Momin Chris Bergeron Management Team Paul Miller Caroline Kurtz Dean Falb Liz Wolffe Aoife Brennen 18

19 19 Back Up

20 Pharmacokinetics of SYNB1618 in mice SYNB1618 exhibits rapid transit GI transit Fecal excretion SYNB1618 gavaged to C57BL/6 mice and GI compartments plated over time Complete clearance from all animals within 48h Transit of SYNB1618 through the small intestine was rapid Progression to primates anticipated to be a more ideal translation model 20