Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Safety and pharmacodynamics of an engineered E. coli Nissle for the treatment of phenylketonuria: a first-in-human phase 1/2a study

Nature Metabolism volume 3, pages 1125–1132 (2021)Cite this article

Subjects

A Publisher Correction to this article was published on 16 August 2022

This article has been updated

Abstract

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

Buy

All prices are NET prices.

Fig. 1: Study design.
Fig. 2: The flow of enrolment in the SAD and MAD parts of the study.
Fig. 3: Plasma TCA production.
Fig. 4: TCA production was observed only in the SYNB1618-treated participants in all SAD and MAD cohorts.
Fig. 5: A dose-responsive, non-saturated increase in urinary HA and D5-HA amount excreted was observed in the H cohorts.
Fig. 6: An increase in the urinary excretion of PLA was observed in HVs treated with SYNB1618 at 1 × 1011 CFU TID but not in the placebo group.

Data availability

Data in the published article (and its Supplementary Information files) have been presented where possible as group-level summaries. Any data presented to illustrate individual patient performance have been de-identified. The data sets generated during and/or analysed during the current study are available from the corresponding author (M.K.P) upon reasonable request including a methodologically sound proposal, although restrictions may apply due to patient privacy and HIPAA regulations.

Change history

References

  1. DeGroot, M. J., Hoeksma, M., Blau, N., Reijngoud, D. J. & Van Spronsen, F. J. Pathogenesis of cognitive dysfunction in phenylketonuria: review of hypotheses. Mol. Genet. Metab. 99, S86–S89 (2010).

    Article  CAS  Google Scholar 

  2. Vockley, J. et al. Phenylalanine hydroxylase deficiency: diagnosis and management guideline. Genet. Med. 16, 188–200 (2014).

    Article  CAS  Google Scholar 

  3. Van Spronsen, F. J. et al. Key European guidelines for the diagnosis and management of patients with phenylketonuria. Lancet Diabetes Endocrinol. 5, 743–756 (2017).

    Article  Google Scholar 

  4. Van Wegberg, A. M. J. et al. The complete European guidelines on phenylketonuria: diagnosis and treatment. Orphanet J. Rare Dis. 12, 162 (2017).

    Article  Google Scholar 

  5. Singh, R. H. et al. Recommendations for the nutrition management of phenylalanine hydroxylase deficiency. Genet. Med. 16, 121–131 (2014).

    Article  CAS  Google Scholar 

  6. Jurecki, E. R. et al. Adherence to clinic recommendations among patients with phenylketonuria in the United States. Mol. Genet. Metab. 120, 190–197 (2017).

    Article  CAS  Google Scholar 

  7. Brown, C. S. & Lichter-Konecki, U. Phenylketonuria (PKU): a problem solved? Mol. Genet. Metab. Rep. 6, 8–12 (2016).

    Article  Google Scholar 

  8. Daelman, L., Sedel, F. & Tourbah, A. Progressive neuropsychiatric manifestations of phenylketonuria in adulthood. Rev. Neurol. 170, 280–287 (2014).

    Article  CAS  Google Scholar 

  9. Bilder, D. A. et al. Systematic review and meta-analysis of neuropsychiatric symptoms and executive functioning in adults with phenylketonuria. Dev. Neuropsychol. 41, 245–260 (2016).

    Article  Google Scholar 

  10. FDA. Kuvan prescribing information. BioMarin Pharmaceutical https://www.accessdata.fda.gov/drugsatfda_docs/label/2014/022181s013lbl.pdf (2014).

  11. Hoskins, J. A. The occurrence, metabolism and toxicity of cinnamic acid and related compounds. J. Appl. Toxicol. 4, 283–292 (1984).

    Article  CAS  Google Scholar 

  12. Kim, W. et al. Trends in enzyme therapy for phenylketonuria. Mol. Ther. 10, 220–224 (2004).

    Article  Google Scholar 

  13. Lindegren, M. L. et al. A systematic review of BH4 (sapropterin) for the adjuvant treatment of phenylketonuria. JIMD Rep. 8, 109–119 (2013).

    Article  Google Scholar 

  14. Vernon, H. J. et al. Introduction of sapropterin dihydrochloride as standard of care in patients with phenylketonuria. Mol. Genet. Metab. 100, 229–233 (2010).

    Article  CAS  Google Scholar 

  15. FDA. Palynziq prescribing information. BioMarin Pharmaceutical https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/761079s000lbl.pdf (2018).

  16. Chang, T. M., Bourget, L. & Lister, C. A new theory of enterorecirculation of amino acids and its use for depleting unwanted amino acids using oral enzyme-artificial cells, as in removing phenylalanine in phenylketonuria. Artif. Cells Blood Substit. Immobil. Biotechnol. 23, 1–21 (1995).

    Article  CAS  Google Scholar 

  17. Sarkissian, C. N. et al. A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase. Proc. Natl Acad. Sci. USA 96, 2339–2344 (1999).

    Article  CAS  Google Scholar 

  18. Levy, H. L., Sarkissian, C. N. & Scriver, C. R. Phenylalanine ammonia lyase (PAL): from discovery to enzyme substitution therapy for phenylketonuria. Mol. Genet. Metab. 24, 223–229 (2018).

    Article  Google Scholar 

  19. Hoskins, J. A. et al. Enzymatic control of phenylalanine intake in phenylketonuria. Lancet 23, 392–394 (1980).

    Article  Google Scholar 

  20. Bourge, L. & Chang, T. M. Effects of oral administration of artificial cells immobilized phenylalanine ammonia-lyase on intestinal amino acids of phenylketonuric rats. Biomater. Artif. Cells Artif. Organs 17, 161–181 (1989).

    Article  Google Scholar 

  21. Fabio Parmeggiani, N., Weise, J., Ahmed, S. T. & Turner, N. J. Synthetic and therapeutic applications of ammonia-lyases and aminomutases. Chem. Rev. 118, 73–118 (2018).

    Article  Google Scholar 

  22. Pereira de Sousa, I., Gourmel, C., Berkovska, O., Burger, M. & Leroux, J.-C. A microparticulate based formulation to protect therapeutic enzymes from proteolytic digestion: phenylalanine ammonia lyase as case study. Nat. Res. Sci. Rep. 10, 3651 (2020).

    Article  CAS  Google Scholar 

  23. Isabella, V. M. et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat. Biotechnol. 36, 857–864 (2018).

    Article  CAS  Google Scholar 

  24. Crook, N. et al. Adaptive strategies of the candidate E. coli Nissle in the mammalian gut. Cell Host Microbe 25, 499–512 (2019).

    Article  CAS  Google Scholar 

  25. Kurtz, C. B. et al. An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans. Sci. Transl. Med. 16, eaau7975 (2019).

    Article  Google Scholar 

  26. Scriver, C. R. & Rosenberg, L. E. Amino Acid Metabolism and its Disorders (Saunders, 1973).

  27. Ardeypharm. Mutaflor Consumer Information https://www.mutaflor.com.au/wp-content/uploads/2014/05/Consumer-Package-Insert-Mutaflor.pdf (2012).

  28. Bioanalytical Method Validation: Guidance for Industry (US Food and Drug Administration, 2018); https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf

  29. R Core Team. R: a Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2020); https://www.R-project.org/

  30. Lenth, R. V. emmeans: Estimated Marginal Means, aka Least-Squares Means. R package v.1.5.0 (2020); https://CRAN.R-project.org/package=emmeans

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

  1. Synlogic, Inc., Cambridge, MA, USA

    Marja K. Puurunen, Larry Blankstein, Mary J. Castillo, Mark R. Charbonneau, Vincent M. Isabella, Vasu V. Sethuraman, Richard J. Riese, Caroline B. Kurtz & Aoife M. Brennan

  2. University of Pittsburgh, Pittsburgh, PA, USA

    Jerry Vockley

  3. UPMC Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA

    Jerry Vockley

  4. PRA Health Sciences, Salt Lake City, UT, USA

    Shawn L. Searle

  5. Boston Children’s Hospital, Boston, MA, USA

    Stephanie J. Sacharow & Benjamin D. Goodlett

  6. Harvard Medical School, Boston, MA, USA

    Stephanie J. Sacharow & Benjamin D. Goodlett

  7. Vanderbilt University Medical Center, Nashville, TN, USA

    John A. Phillips III

  8. Human Predictions LLC, Boston, MA, USA

    William S. Denney

  9. Metabolic Solutions, Nashua, NH, USA

    David A. Wagner

Authors
  1. Marja K. Puurunen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jerry Vockley
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shawn L. Searle
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephanie J. Sacharow
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. John A. Phillips III
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. William S. Denney
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Benjamin D. Goodlett
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. David A. Wagner
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Larry Blankstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Mary J. Castillo
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Mark R. Charbonneau
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Vincent M. Isabella
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Vasu V. Sethuraman
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Richard J. Riese
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Caroline B. Kurtz
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Aoife M. Brennan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization and design of the work were carried out by M.K.P., W.S.D., L.B., V.M.I., C.B.K. and A.M.B. The acquisition of data was performed by M.K.P., L.B., J.V., S.L.S, S.J.S. and J.A.P. The analysis and interpretation of data were performed by M.K.P., W.S.D., B.D.G., D.A.W., M.J.C., M.R.C., R.J.R., C.B.K. and A.M.B. Trial delivery and administration were carried out by L.B. and V.V.S. The original draft of the manuscript was written by M.K.P., W.S.D., V.V.S., M.J.C., M.R.C., R.J.R., C.B.K. and A.M.B. The submitted paper was reviewed and approved by all the authors.

Corresponding author

Correspondence to Marja K. Puurunen.

Ethics declarations

Competing interests

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.

Additional information

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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 SYNB1618 Mechanism of Action.

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.

Supplementary information

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s42255-021-00430-7

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing