Fig. 1 Characterization of SYNB1020. (A) Schematic of the engineered strain SYNB1020, including its intended effect on l-arg metabolism. (B) l-arg production by engineered strains in vitro. Data represent measurements from three independent bacterial cultures ± SD. P values described in the text were generated using two-tailed unpaired Student’s t test. (C) Strain activity was tested in modified M9 medium supplemented with 5 mM ammonia. Assay reactions contained chloramphenicol (10 μg/ml) to inhibit ammonia consumption due to cell division and de novo protein synthesis. SYNB1020 cells were induced by incubation at low oxygen (EcNStR was treated in a similar manner). Ammonia consumption by EcNStR and SYNB1020 was quantified using the indophenol-blue method. (D) Arginine production by the same samples in (C) was quantified using liquid chromatography–tandem mass spectrometry after derivatization with dansyl-chloride. Data are representative of triplicate measurements ± SD. (E) Volcano plot of gene expression in SYNB1020 as compared to EcN, under microaerobic conditions in the presence of thymidine. Genes with an adjusted P value (Padj) of less than 0.1, as reported by DESeq2, were considered differentially expressed. ArgB, acetyl glutamate kinase; ArgC, N-acetyl glutamylphosphate reductase; ArgD, N-acetylornithine aminotransferase; ArgE, acetylornithine deacetylase; ArgFI, ornithine carbamoyltransferase; ArgG, arginosuccinate synthase; ArgH, arginosuccinate lyase; CarAB, carbamoyl phosphate synthetase; fbr, feedback resistant; FNR, “fumarate and nitrate reduction” transcription regulator; Gln, glutamine; Glu, glutamate; HCO3−, bicarbonate; KG, ketoglutarate; malEK, intergenic region between malE and malK genes; ΔthyA, thymidylate synthase deletion.
Fig. 2 In vivo activity of engineered strains. (A) Blood ammonia and percent survival in OTCspf-ash mice receiving normal chow or high-protein diet and dosed orally (PO) with vehicle [15% glycerol in phosphate-buffered saline (PBS)], heat-killed SYNB1020 (dead) or 1 × 109, 5 × 109, or 1 × 1010 colony-forming units (CFU) of SYNB1020 (n = 5 vehicle/chow, n = 5 SYNB1020 dead/high protein, and n = 9 per group for all other groups). Statistical analysis comparing chow/vehicle with high-protein/SYNB1020 dead was performed using unpaired Student’s t test. Statistical analysis comparing high-protein/SYNB1020 dead with high-protein/SYNB1020KR was performed using one-way ANOVA on ranks, followed by Dunnett’s multiple comparison test. (B) Top: Blood ammonia concentrations in OTC spf ash mice receiving normal chow or high-protein diet and dosed PO with vehicle (H2O), EcNΔargRΔthyA (1 × 1010 CFU) or SYNB1020KR (1 × 1010 CFU) for 9 days (n = 5 vehicle/chow, n = 10 vehicle/high-protein, n = 7 EcNΔargRΔthyA, and n = 10 SYNB1020KR). Statistical analysis comparing chow/vehicle with high-protein/vehicle was performed using unpaired Student’s t test. Statistical analysis comparing high-protein/vehicle with high-protein/EcNΔargRΔthyA or SYNB1020 KR was performed using one-way ANOVA, followed by Dunnett’s multiple comparison test. Bottom: Survival of OTC spf ash mice receiving normal chow or high-protein diet and dosed PO with vehicle, EcNΔargRΔthyA or SYNB1020KR for 9 days. Statistical analysis was performed using a logl-rank (Mantel-Cox) test. (C) Blood ammonia in Balb/cJ mice intraperitoneally (IP) dosed with or without TAA (150 mg/kg) in combination with vehicle (15% glycerol in PBS), were dosed PO with EcN (1 × 1010 CFU), or SYNB1020 (1 × 1010 CFU) for 3 weeks. Blood ammonia was measured on the last day of the experiment (n = 10 per group for vehicle, EcN/TAA, and SYNB1020/TAA and n = 9 for vehicle/TAA). Statistical analysis comparing vehicle with TAA/vehicle was performed using unpaired Student’s t test with Welch’s correction. Statistical analysis comparing TAA/vehicle with TAA/EcN or TAA/SYNB1020 was performed using one-way ANOVA, followed by Tukey’s multiple comparison test. Data are presented as means ± SEM. Statistical significance indicated as *P < 0.05, **P < 0.01, and ***P < 0.0001.
Fig. 3 SYNB1020 excretion profile. SYNB1020 excretion profile in (A) female (n = 16 per group) or (B) male (n = 16 per group) CD1 mice dosed with vehicle, 5 × 109 or 1.5 × 1011 CFU SYNB1020 for 28 days. Fecal pellets were sterilely collected and analyzed for the presence of SYNB1020 DNA by qPCR using a validated method with a LLOQ of 10 copies/5 ng of DNA.
Fig. 4 Evaluation of SYNB1020 safety and pharmacodynamics in healthy volunteers receiving 14 days of dosing with SYNB1020 or placebo. (A) Diurnal mean plasma ammonia levels measured over 24 hours across MAD cohorts of healthy volunteers treated with placebo (n = 6), 2 × 109 CFU TID (n = 6), 2 × 1011 CFU TID (n = 6), or 5 × 1011 CFU TID (n = 5). (B) Individual 24-hour plasma AUC ammonia concentrations in MAD cohorts treated with placebo (n = 6), 2 × 109 CFU TID (n = 6), 2 × 1011 CFU TID (n = 6), or 5 × 1011 CFU TID (n = 5). Individual volunteers are shown as black lines. Mean ammonia AUC per cohort is depicted in blue. (C) Urinary nitrate concentrations were measured 24 hours after dose in MAD cohorts by a colorimetric method in MAD cohorts treated with placebo (n = 6), 2 × 109 CFU TID (n = 6), 2 × 1011 CFU TID (n = 6), or 5 × 1011 CFU TID (n = 5). Nitrate concentration was multiplied by the total urine volume to obtain total nitrate in micromole. (D) Mean plasma AUC (0 to 24 hours) 15N-nitrate change from baseline (CFB) after oral administration of 15N-NH3Cl in MAD cohorts treated with placebo (n = 6), 2 × 109 CFU TID (n = 6), 2 × 1011 CFU TID (n = 6), or 5 × 1011 CFU TID (n = 5). (E) Total 15N-urinary nitrate change from baseline after oral administration of 15N-NH3Cl in MAD cohorts treated with placebo (n = 6), 2 × 109 CFU TID (n = 6), 2 × 1011 CFU TID (n = 6), or 5 × 1011 CFU TID (n = 5).
Fig. 5 SYNB1020 excretion profile and activity in stool from healthy volunteers. Groups of eight volunteers per cohort (six SYNB1020 and two placebo) were enrolled and dosed with either placebo (n = 6) or SYNB1020 at 2 × 109 CFU TID (n = 6), 2 × 1011 CFU TID (n = 6), or 5 × 1011 CFU TID (n = 5). (A) Steady-state concentrations of SYNB1020 DNA in feces of healthy volunteers dosed for 14 days. (B) Clearance of SYNB1020 DNA from volunteers’ stool after dosing using qPCR (LLOQ, 10 copies/5 ng of DNA). (C) 15N4–l-arg production after normal stool (n = 2 per group) was spiked with 15N-NH3 and increasing doses of SYNB1020. (D) Stool from healthy volunteers dosed with placebo (black circles; n = 2 per group) or SYNB1020 (open squares; n = 6 per group) was inoculated with 15N-NH3, and 15N4–l-arg was measured.
- Table 1 Bacterial strains.
EcN was genetically engineered with the described series of changes to create SYNB1020 and related tool strains that we used to elucidate activity in in vitro and in vivo studies.
Strain Genotype EcN E. coli Nissle 1917 EcNStrR EcN, streptomycin resistant EcNKR EcN, kanamycin resistant EcNΔargR EcN, ΔargR EcNΔthyA EcN, ΔthyA::CmR EcNargAfbr+ EcN, malEK::CmR-PfnrS-argAfbr EcNΔthyA argAfbr+ EcN, ΔthyA, malEK::CmR-PfnrS-argAfbr EcNΔargR ΔthyA EcN, ΔargR, ΔthyA::CmR EcNΔthyA, argAfbr+ EcN, ΔthyA, malEK::CmR-PfnrS-argAfbr EcN-GFP EcN containing plasmid with PfnrS–
GFP (green fluorescent protein)
transcription fusionSYNB1020thyA+ EcN, ΔargR, malEK::PfnrS-argAfbr SYNB1020 EcN, ΔargR, ΔthyA, malEK::PfnrS-argAfbr SYNB1020KR EcN, ΔargR, ΔthyA, malEK::PfnrS-argAfbrKanR - Table 2 Treatment-emergent adverse events.
Adverse events reported by healthy volunteers dosed for either a single or multiple (14 days) days of dosing with SYNB1020. All reported adverse events were of mild or moderate severity. QD, once daily; TEAE, treatment-emergent adverse event.
System organ
class
preferred termSingle-day cohorts Multiple-day cohorts Pooled placebo
cohorts2 × 109
CFU QD
(N = 3)
n (%)2 × 1010
CFU QD
(N = 3)
n (%)2 × 1011
CFU QD
(N = 3)
n (%)2 × 1012
CFU QD
(N = 3)
n (%)5 × 1011
CFU TID
(N = 3)
n (%)1 × 1012
CFU TID
(N = 3)
n (%)2 × 1012
CFU TID
(N = 3)
n (%)2 × 109
CFU TID
(N = 6)
n (%)2 × 1011
CFU TID
(N = 6)
n (%)5 × 1011
CFU TID
(N = 6)
n (%)SAD
part
(N = 7)
n (%)MAD
part
(N = 6)
n (%)Any TEAE 0 0 0 2 (67) 1 (33) 1 (33) 2 (67) 0 5 (83) 2 (33) 0 1 (17) GI disorders 0 0 0 2 (67) 0 1 (33) 2 (67) 0 3 (50) 2 (33) 0 1 (17) Nausea 0 0 0 1 (33) 0 1 (33) 2 (67) 0 2 (33) 2 (33) 0 0 Abdominal
pain0 0 0 1 (33) 0 0 0 0 2 (33) 1 (17) 0 0 Vomiting 0 0 0 0 0 1 (33) 2 (67) 0 0 1 (17) 0 0 Abdominal
discomfort0 0 0 0 0 0 0 0 1 (17) 1 (17) 0 0 Abdominal
distension0 0 0 1 (33) 0 0 0 0 0 0 0 0 Diarrhea 0 0 0 0 0 0 0 0 0 1 (17) 0 0 Flatulence 0 0 0 0 0 0 0 0 0 0 0 1 (17) General disorders
and
administration
site conditions0 0 0 2 (67) 0 0 0 0 2 (33) 1 (17) 0 1 (17) Catheter site
swelling0 0 0 2 (67) 0 0 0 0 0 0 0 1 (17) Vessel
puncture site
pain0 0 0 1 (33) 0 0 0 0 0 1 (17) 0 0 Catheter site
bruise0 0 0 0 0 0 0 0 1 (17) 0 0 0 Catheter site
pain0 0 0 1 (33) 0 0 0 0 0 0 0 0 Vessel
puncture site
bruise0 0 0 0 0 0 0 0 1 (17) 0 0 0 Infections and
infestations0 0 0 0 1 (33) 0 0 0 0 0 0 0 Upper
respiratory
tract infection0 0 0 0 1 (33) 0 0 0 0 0 0 0 Nervous system
disorders0 0 0 0 0 0 0 0 0 1 (17) 0 0 Headache 0 0 0 0 0 0 0 0 0 1 (17) 0 0
Supplementary Materials
www.sciencetranslationalmedicine.org/cgi/content/full/11/475/eaau7975/DC1
Materials and Methods
Fig. S1. Demonstration of in vivo induction of the PfnrS using green fluorescent protein as a reporter in EcN strain.
Fig. S2. EcN strains lacking the thyA gene fail to grow in rich media lacking exogenous thymidine.
Fig. S3. Differential gene expression for SYNB1020 compared to EcN for genes in the l-arg biosynthesis pathway.
Fig. S4. Dose-dependent fecal excretion of EcN and SYNB1020 strains in mice.
Fig. S5. Study design of a randomized, double-blinded, placebo-controlled study to assess the safety, tolerability, and pharmacodynamics of SYNB1020 in healthy volunteers.
Fig. S6. Change from baseline in C-reactive protein.
Fig. S7. Time-matched change from baseline for diastolic blood pressure.
Fig. S8. Time-matched change from baseline for systolic blood pressure.
Table S1. Differential gene expression comparisons of SYNB1020 and EcNΔthyA to EcN in vitro.
Table S2. Quantification of viable SYNB1020KR in mouse cecolonic samples over time.
Table S3. Average ECNStrR or SYNB1020KR CFU recovered from GI contents of mice dosed with each strain.
Table S4. Activity rate of SYNB1020KR recovered from mouse cecum and colon.
Table S5. Arginine pathway metabolites in mouse and nonhuman primates.
Table S6. Clinical and laboratory results from a 28-day oral toxicity study of SYNB1020 in mice.
Table S7. Mice with detectable SYNB1020 DNA in tissues as measured by qPCR.
Table S8. Baseline demographics for the phase 1 study population.
Table S9. ECG QT interval in the phase 1, multiple-day, study population.
Table S10. Laboratory results for urea cycle products in the multiple-day cohort phase 1 study population.
Table S11. 15N metabolites in the phase 1 study population.
Table S12. Primers used to characterize SYNB1020 genetic modifications.
Table S13. Fermentation medium (FM1).
References (81–90)
Additional Files
The PDF file includes:
- Materials and Methods
- Fig. S1. Demonstration of in vivo induction of the PfnrS using green fluorescent protein as a reporter in EcN strain.
- Fig. S2. EcN strains lacking the thyA gene fail to grow in rich media lacking exogenous thymidine.
- Fig. S3. Differential gene expression for SYNB1020 compared to EcN for genes in the
l -arg biosynthesis pathway. - Fig. S4. Dose-dependent fecal excretion of EcN and SYNB1020 strains in mice.
- Fig. S5. Study design of a randomized, double-blinded, placebo-controlled study to assess the safety, tolerability, and pharmacodynamics of SYNB1020 in healthy volunteers.
- Fig. S6. Change from baseline in C-reactive protein.
- Fig. S7. Time-matched change from baseline for diastolic blood pressure.
- Fig. S8. Time-matched change from baseline for systolic blood pressure.
- Legend for table S1
- Table S2. Quantification of viable SYNB1020KR in mouse cecolonic samples over time.
- Table S3. Average ECNStrR or SYNB1020KR CFU recovered from GI contents of mice dosed with each strain.
- Table S4. Activity rate of SYNB1020KR recovered from mouse cecum and colon.
- Table S5. Arginine pathway metabolites in mouse and nonhuman primates.
- Table S6. Clinical and laboratory results from a 28-day oral toxicity study of SYNB1020 in mice.
- Table S7. Mice with detectable SYNB1020 DNA in tissues as measured by qPCR.
- Table S8. Baseline demographics for the phase 1 study population.
- Table S9. ECG QT interval in the phase 1, multiple-day, study population.
- Table S10. Laboratory results for urea cycle products in the multiple-day cohort phase 1 study population.
- Table S11. 15N metabolites in the phase 1 study population.
- Table S12. Primers used to characterize SYNB1020 genetic modifications.
- Table S13. Fermentation medium (FM1).
- References (81–90)
Other Supplementary Material for this manuscript includes the following:
- Table S1 (Microsoft Excel format). Differential gene expression comparisons of SYNB1020 and EcNΔthyA to EcN in vitro.
