Phenylketonuria (PKU) is a rare disease caused by biallelic mutations in the PAH gene that result in an inability to convert phenylalanine (Phe) to tyrosine, elevated blood Phe levels and severe neurological complications if untreated. Most patients are unable to adhere to the protein-restricted diet, and thus do not achieve target blood Phe levels. We engineered a strain of E. coli Nissle 1917, designated SYNB1618, through insertion of the genes encoding phenylalanine ammonia lyase and l-amino acid deaminase into the genome, which allow for bacterial consumption of Phe within the gastrointestinal tract. SYNB1618 was studied in a phase 1/2a randomized, placebo-controlled, double-blind, multi-centre, in-patient study (NCT03516487) in adult healthy volunteers (n = 56) and patients with PKU and blood Phe level ≥600 mmol l−1 (n = 14). Participants were randomized to receive a single dose of SYNB1618 or placebo (part 1) or up to three times per day for up to 7 days (part 2). The primary outcome of this study was safety and tolerability, and the secondary outcome was microbial kinetics. A D5-Phe tracer (15 mg kg−1) was used to study exploratory pharmacodynamic effects. SYNB1618 was safe and well tolerated with a maximum tolerated dose of 2 × 1011 colony-forming units. Adverse events were mostly gastrointestinal and of mild to moderate severity. All participants cleared the bacteria within 4 days of the last dose. Dose-responsive increases in strain-specific Phe metabolites in plasma (trans-cinnamic acid) and urine (hippuric acid) were observed, providing a proof of mechanism for the potential to use engineered bacteria in the treatment of rare metabolic disorders.
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We are grateful to the study patients and their families, and to the research nurses and study staff, as well as the National PKU Association for all their support. We thank N. Longo and C. Harding for their invaluable scientific advice. We greatly appreciate the technical and editorial support provided by J. Blasbalg, C. Anderson, P. Cantarella, M. James, M. Daza and K. Pace. Funding for this study was provided by Synlogic, Inc. The funders participated in study design, data analysis and preparation of the manuscript.
J.V. reports research funding from Biomarin Pharmaceuticals, Homology Pharmaceuticals, Nestle, PTC Therapeutics, outside the submitted work. M.K.P., L.B., V.V.S., V.M.I., M.J.C., M.R.C., R.J.R., C.B.K. and A.M.B. are full-time employees and hold equity in Synlogic Inc. D.A.W. receives a salary from Metabolic Solutions. Metabolic Solutions was paid a fee to analyse samples for this publication. W.S.D. is a consultant for Synlogic Inc. The remaining authors declare no competing interests.
Peer review information Nature Metabolism thanks Nicole Mayer Hamblett and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: George Caputa.
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SYNB1618 contains chromosomally inserted genes encoding PheP, a high-affinity Phe transporter that can bring Phe into the cell cytoplasm, PAL, which converts Phe to trans-cinnamate, and LAAD, which converts Phe to phenylpyruvate. Induction of these components is carried out partially by the anaerobic-responsive transcriptional activator FNR, for strain activation of PAL and pheP in the anoxic environment of the mammalian gut. Additional copies of PAL and LAAD are placed under control of the Isopropyl β-D-1-thiogalactopyranoside, and L-arabinose inducible promoters, respectively, for strain activation in vitro during production of drug product. Abbreviations: AraC = arabinose-responsive transcriptional regulator; FNR = fumarate and nitrate reductase regulator; LAAD = L-amino acid deaminase from Proteus mirabilis; PAL = phenylalanine ammonia lyase from Photorhabdus luminescens; PheP = high-affinity phenylalanine transporter; pheP = gene encoding high-affinity phenylalanine transporter; Ptac = synthetic promoter controlling PAL expression and regulated by the LacI transcriptional repressor; ΔdapA = deletion of dapA gene leading to diaminopimelate auxotrophy.
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Puurunen, M.K., Vockley, J., Searle, S.L. et al. Safety and pharmacodynamics of an engineered E. coli Nissle for the treatment of phenylketonuria: a first-in-human phase 1/2a study. Nat Metab 3, 1125–1132 (2021). https://doi.org/10.1038/s42255-021-00430-7