Review Article | Published:

Engineering bacteria for diagnostic and therapeutic applications

Nature Reviews Microbiology volume 16, pages 214225 (2018) | Download Citation

Abstract

Our ability to generate bacterial strains with unique and increasingly complex functions has rapidly expanded in recent times. The capacity for DNA synthesis is increasing and costing less; new tools are being developed for fast, large-scale genetic manipulation; and more tested genetic parts are available for use, as is the knowledge of how to use them effectively. These advances promise to unlock an exciting array of 'smart' bacteria for clinical use but will also challenge scientists to better optimize preclinical testing regimes for early identification and validation of promising strains and strategies. Here, we review recent advances in the development and testing of engineered bacterial diagnostics and therapeutics. We highlight new technologies that will assist the development of more complex, robust and reliable engineered bacteria for future clinical applications, and we discuss approaches to more efficiently evaluate engineered strains throughout their preclinical development.

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References

  1. 1.

    The treatment of malignant tumors by repeated inoculations of erysipelas: with a report of ten original cases. Am. J. Med. Sci. 105, 487–510 (1893).

  2. 2.

    Aus der Sitzung der medicinischen section vom 13. November 1867 [GERMAN]. Berl. Klin. Wochenschr. 5, 137 (1868).

  3. 3.

    Ueber die Züchtung der Erysipelkokken auf künstlichem Nährboden und ihre Übertragbarkeit auf den Menschen [GERMAN]. Dtsch. Med. Wochenschr. 8, 553–554 (1882).

  4. 4.

    et al. Reversal of diabetes in NOD mice by clinical-grade proinsulin and IL-10-secreting Lactococcus lactisin combination with low-dose anti-CD3 depends on the induction of Foxp3-positive T cells. Diabetes 66, 448–459 (2017).

  5. 5.

    et al. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease. Clin. Gastroenterol. Hepatol. 4, 754–759 (2006).

  6. 6.

    et al. Prevention of vaginal SHIV transmission in macaques by a live recombinant Lactobacillus. Mucosal Immunol. 4, 648–657 (2011).

  7. 7.

    et al. Two-step enhanced cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous flagellin. Sci. Transl Res. 9, eaak9537 (2017).

  8. 8.

    et al. Oral delivery of IL-27 recombinant bacteria attenuates immune colitis in mice. Gastroenterology 146, 210–221.e213 (2014). A nice demonstration of the unique capabilities of live bacteria over recombinant protein delivery using L. lactis expressing IL-27 to reduce colitis in the mouse.

  9. 9.

    et al. Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models. Nat. Commun. 8, 15028 (2017).

  10. 10.

    , , , & Antimicrobial probiotics reduce Salmonella enterica in turkey gastrointestinal tracts. Sci. Rep. 7, 40695 (2017).

  11. 11.

    Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289, 1352–1355 (2000). A classic paper of the field, describing the use of IL-10 secreting L. lactis bacteria to mitigate colitis in mice.

  12. 12.

    et al. Food-grade bacteria expressing elafin protect against inflammation and restore colon homeostasis. Sci. Transl Med. 4, 158ra144 (2012).

  13. 13.

    et al. Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice. Gastroenterology 127, 502–513 (2004).

  14. 14.

    et al. Orally administered L. lactis secreting an anti-TNF nanobody demonstrate efficacy in chronic colitis. Mucosal Immunol. 3, 49–56 (2010).

  15. 15.

    et al. Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine. Infect. Immun. 66, 3183–3189 (1998).

  16. 16.

    , & Attenuated Shigella as a DNA delivery vehicle for DNA-mediated immunization. Science 270, 299–302 (1995).

  17. 17.

    , , & Induction of a cellular immune response to a foreign antigen by a recombinant Listeria monocytogenes vaccine. J. Immunol. 149, 53–59 (1992).

  18. 18.

    et al. Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation. Nat. Biotechnol. 35, 653–658 (2017). Long-term testing of a bacterial diagnostic for inflammation in mice. Circuit function and sequence was tested during >6 months of constant colonization of the gut.

  19. 19.

    et al. Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Mol. Syst. Biol. 13, 923 (2017).

  20. 20.

    et al. Phase 1b, multicenter, single blinded, placebo-controlled, sequential dose escalation study to assess the safety and tolerability of topically applied AG013 in subjects with locally advanced head and neck cancer receiving induction chemotherapy. Cancer 119, 4268–4276 (2013).

  21. 21.

    et al. Safety and survival with GVAX pancreas prime and Listeria monocytogenes–expressing mesothelin (CRS-207) boost vaccines for metastatic pancreatic cancer. J. Clin. Oncol. 33, 1325–1333 (2015).

  22. 22.

    , & The first clinical use of a live-attenuated Listeria monocytogenes vaccine: a phase i safety study of Lm-LLO-E7 in patients with advanced carcinoma of the cervix. Vaccine 27, 3975–3983 (2009).

  23. 23.

    , , , & Attenuated Listeria monocytogenes vaccine vectors expressing Influenza A nucleoprotein: preclinical evaluation and oral inoculation of volunteers. Microbiol. Immunol. 55, 304–317 (2011).

  24. 24.

    et al. Safety and shedding of an attenuated strain of Listeria monocytogenes with a deletion of actA/plcB in adult volunteers: a dose escalation study of oral inoculation. Infect. Immun. 70, 3592–3601 (2002).

  25. 25.

    et al. A live-attenuated Listeria vaccine (ANZ-100) and a live-attenuated Listeria vaccine expressing mesothelin (CRS-207) for advanced cancers: phase I studies of safety and immune induction. Clin. Cancer Res. 18, 858–868 (2012).

  26. 26.

    et al. Results from a phase 2b, randomized, multicenter study of GVAX pancreas and CRS-207 compared to chemotherapy in adults with previously-treated metastatic pancreatic adenocarcinoma (ECLIPSE Study). J. Clinl Oncol. 35, 345–345 (2017).

  27. 27.

    et al. Digital-to-biological converter for on-demand production of biologics. Nat. Biotechnol. 29, 544 (2017). An insight into the possible future of automation in the construction and testing of biologics.

  28. 28.

    et al. Genetic circuit design automation. Science 352, aac734 (2016).

  29. 29.

    , & Short hairpin RNA-expressing bacteria elicit RNA interference in mammals. Nat. Biotechnol. 24, 697–702 (2006).

  30. 30.

    et al. Attenuated Salmonella typhimurium containing interleukin-2 decreases MC-38 hepatic metastases: a novel anti-tumor agent. Cancer. Biother. Radiopharm. 11, 145–153 (1996).

  31. 31.

    et al. Tumor amplified protein expression therapy: Salmonella as a tumor-selective protein delivery vector. Oncol. Res. 12, 127–135 (2000).

  32. 32.

    et al. Two Listeria monocytogenes vaccine vectors that express different molecular forms of human papilloma virus-16 (HPV-16) E7 induce qualitatively different T cell immunity that correlates with their ability to induce regression of established tumors immortalized by HPV-16. J. Immunol. 167, 6471–6479 (2001).

  33. 33.

    , , & Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis. Gene Ther. 9, 291–296 (2002).

  34. 34.

    et al. In vivo antigen delivery by a Salmonella typhimurium type III secretion system for therapeutic cancer vaccines. J. Clin. Invest. 116, 1946–1954 (2006).

  35. 35.

    , , & Attenuated Salmonella engineered to produce human cytokine LIGHT inhibit tumor growth. Proc. Natl Acad. Sci. USA 104, 12879–12883 (2007).

  36. 36.

    et al. Bacterial delivery of a novel cytolysin to hypoxic areas of solid tumors. Gene Ther. 16, 329–339 (2009).

  37. 37.

    et al. Synchronized cycles of bacterial lysis for in vivo delivery. Nature 536, 81–85 (2016).

  38. 38.

    et al. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat. Biotechnol. 21, 785–789 (2003).

  39. 39.

    et al. Paracellular entry of interleukin-10 producing Lactococcus lactis in inflamed intestinal mucosa in mice. Inflamm. Bowel Dis. 14, 471–479 (2008). Development and testing of several technological features that underpin several ongoing L. lactis therapeutic efforts, including the use of thiamine deficiency as a biocontainment measure.

  40. 40.

    et al. In vivo mucosal delivery of bioactive human interleukin 1 receptor antagonist produced by Streptococcus gordonii. BMC Biotechnol. 3, 15 (2003).

  41. 41.

    , , , & Mucosal delivery of anti-inflammatory IL-1Ra by sporulating recombinant bacteria. BMC Biotechnol. 4, 27 (2004).

  42. 42.

    et al. Treatment of colitis with a commensal gut bacterium engineered to secrete human TGF-beta1 under the control of dietary xylan 1. Inflamm. Bowel Dis. 17, 1925–1935 (2011).

  43. 43.

    et al. A transgenic probiotic secreting a parasite immunomodulator for site-directed treatment of gut inflammation. Mol. Ther. 22, 1730–1740 (2014).

  44. 44.

    et al. AG013, a mouth rinse formulation of Lactococcus lactis secreting human Trefoil Factor 1, provides a safe and efficacious therapeutic tool for treating oral mucositis. Oral Oncol. 46, 564–570 (2010).

  45. 45.

    et al. In situ delivery of passive immunity by lactobacilli producing single-chain antibodies. Nat. Biotechnol. 20, 702–706 (2002).

  46. 46.

    , & Engineered commensal bacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes. Diabetes 64, 1794–1803 (2015).

  47. 47.

    et al. Reversal of autoimmune diabetes by restoration of antigen-specific tolerance using genetically modified Lactococcus lactis in mice. J. Clin. Invest. 122, 1717–1725 (2012).

  48. 48.

    et al. Oral delivery of glutamic acid decarboxylase (GAD)-65 and IL10 by Lactococcus lactis reverses diabetes in recent-onset NOD mice. Diabetes 63, 2876–2887 (2014).

  49. 49.

    , , , & Oral delivery of glucagon like peptide-1 by a recombinant Lactococcus lactis. Pharm. Res. 31, 3404–3414 (2014).

  50. 50.

    et al. Oral delivery of pentameric glucagon-like peptide-1 by recombinant Lactobacillus in diabetic rats. PLOS ONE 11, e0162733 (2016).

  51. 51.

    , & A new biological agent for treatment of Shiga toxigenic Escherichia coli infections and dysentery in humans. Nat. Med. 6, 265–270 (2000).

  52. 52.

    , , , & A recombinant probiotic for treatment and prevention of cholera. Gastroenterology 130, 1688–1695 (2006).

  53. 53.

    & Engineered bacterial communication prevents Vibrio cholerae virulence in an infant mouse model. Proc. Natl Acad. Sci. USA 107, 11260–11264 (2010).

  54. 54.

    et al. Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide. Proc. Natl Acad. Sci. USA 102, 11993–11998 (2005).

  55. 55.

    et al. Engineered vaginal lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-N. Antimicrob. Agents Chemother. 50, 3250–3259 (2006).

  56. 56.

    et al. Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity. J. Clin. Invest. 124, 3391–3406 (2014).

  57. 57.

    et al. An intranasal administration of Lactococcus lactis strains expressing recombinant interleukin-10 modulates acute allergic airway inflammation in a murine model. Clin. Exp. Allergy 40, 1541–1551 (2010).

  58. 58.

    et al. Effective treatment of hypertension by recombinant Lactobacillus plantarum expressing angiotensin converting enzyme inhibitory peptide. Microb. Cell Fact. 14, 202 (2015).

  59. 59.

    Discovering and improving novel peptide therapeutics. Curr. Opin. Pharmacol. 8, 616–619 (2008).

  60. 60.

    & Biology of Incretins: GLP-1 and GIP. Gastroenterology 132, 2131–2157 (2007).

  61. 61.

    , & Glucagon-like peptide 1 (1–37) converts intestinal epithelial cells into insulin-producing cells. Proc. Natl Acad. Sci. USA 100, 5034–5039 (2003).

  62. 62.

    , & The turnover and shedding of epithelial cells. I. The turnover in the gastrointestinal tract. Gut 2, 110–118 (1961).

  63. 63.

    & Vaccination strategies to promote mucosal antibody responses. Immunity 33, 479–491 (2010).

  64. 64.

    , , , & 64 000 Mr autoantibodies as predictors of insulin-dependent diabetes. Lancet 335, 1357–1360 (1990).

  65. 65.

    et al. Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library. Nat. Med. 5, 1026–1031 (1999).

  66. 66.

    & Mechanisms of immune tolerance to allergens: role of IL-10 and Tregs. J. Clin. Invest. 124, 4678–4680 (2014).

  67. 67.

    Immune responses to Listeria monocytogenes. Nat. Rev. Immunol. 4, 812–823 (2004).

  68. 68.

    et al. Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J. Clin. Oncol. 20, 142–152 (2002).

  69. 69.

    et al. Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients. Cancer Gene Ther. 10, 737–744 (2003).

  70. 70.

    et al. Trial watch. Oncoimmunology 3, e29030 (2014).

  71. 71.

    et al. Recombinant human interleukin 10 in the treatment of patients with mild to moderately active Crohn's disease. The Interleukin 10 Inflammatory Bowel Disease Cooperative Study Group. Gastroenterology 119, 1473–1482 (2000).

  72. 72.

    et al. Interleukin 10 (Tenovil) in the prevention of postoperative recurrence of Crohn's disease. Gut 49, 42–46 (2001).

  73. 73.

    et al. Treatment of Crohn's disease with recombinant human interleukin 10 induces the proinflammatory cytokine interferon gamma. Gut 50, 191–195 (2002).

  74. 74.

    Evaluate. Press release. ActoGenix completes phase 2A clinical trial of AG011. Evaluate (2009).

  75. 75.

    et al. A dominant function for interleukin 27 in generating interleukin 10-producing anti-inflammatory T cells. Nat. Immunol. 8, 1380–1389 (2007).

  76. 76.

    et al. Interleukins 27 and 6 induce STAT3-mediated T cell production of interleukin 10. Nat. Immunol. 8, 1363–1371 (2007).

  77. 77.

    , , & Models for drug absorption from the small intestine: where are we and where are we going? Drug Discov. Today, 22, 761–775 (2017).

  78. 78.

    et al. Specific high affinity binding of human interleukin 1β by Caf1A usher protein of Yersinia pestis. FEBS Lett. 371, 65–68 (1995).

  79. 79.

    , , , & Tumor necrosis factor alpha binding to bacteria: evidence for a high-affinity receptor and alteration of bacterial virulence properties. Infect. Immun. 61, 830–835 (1993).

  80. 80.

    Recognition of host immune activation by Pseudomonas aeruginosa. Science 309, 774–777 (2005).

  81. 81.

    , , , & The QseC sensor kinase: a bacterial adrenergic receptor. Proc. Natl Acad. Sci. USA 103, 10420–10425 (2006).

  82. 82.

    , & A bacterial high-affinity GABA binding protein: isolation and characterization. Biochem. Biophys. Res. Commun. 268, 65–68 (2000).

  83. 83.

    , , , & Tunable thermal bioswitches for in vivo control of microbial therapeutics. Nat. Chem. Biol. 13, 75–80 (2016).

  84. 84.

    et al. Rational design of evolutionarily stable microbial kill switches. Mol. Cell 68, 686–697.e3 (2017).

  85. 85.

    et al. Rapid fucosylation of intestinal epithelium sustains host–commensal symbiosis in sickness. Nature 514, 638–641 (2014).

  86. 86.

    et al. Gut inflammation provides a respiratory electron acceptor for Salmonella. Nature 467, 426–429 (2010).

  87. 87.

    , , , & Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates. Sci. Transl Med. 7, 289ra283 (2015). An interesting approach using engineered bacteria as diagnostics grown on ex vivo human samples.

  88. 88.

    et al. Programmable bacteria detect and record an environmental signal in the mammalian gut. Proc. Natl Acad. Sci. USA 111, 4838–4843 (2014). The first test of a synthetic memory circuit in vivo in a mouse model, as well as isolation of a mouse commensal E. coli strain capable of long-term competitive growth in the mouse without selection by antibiotics.

  89. 89.

    et al. Programmable probiotics for detection of cancer in urine. Sci. Transl Med. 7, 289ra284 (2015).

  90. 90.

    et al. Microbiology of bacterial translocation in humans. Gut 42, 29–35 (1998).

  91. 91.

    , , & Genetically modified bacteria as a tool to detect microscopic solidtumour masses with triggered release of a recombinant biomarker. Integr. Biol. 7, 423–434 (2015).

  92. 92.

    & Bacterially speaking. Cell 125, 237–246 (2006).

  93. 93.

    , & Genetically programmable pathogen sense and destroy. ACS Synth. Biol. 2, 715–723 (2013).

  94. 94.

    et al. Reprogramming microorganisms to be pathogen-seeking killers. ACS Synth. Biol. 3, 228–237 (2014).

  95. 95.

    et al. Engineering microorganisms to sense and eradicate Pseudomonas aeruginosa, a human pathogen. Mol. Syst. Biol. 7, 521–521 (2011).

  96. 96.

    , , & Modified lactic acid bacteria detect and inhibit multiresistant enterococci. ACS Synth. Biol. 4, 299–306 (2015).

  97. 97.

    , & Quorum-sensing Salmonella selectively trigger protein expression within tumours. Proc. Natl Acad. Sci. USA 112, 3457–3462 (2015).

  98. 98.

    et al. In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit. Nat. Methods 4, 937–942 (2007).

  99. 99.

    Committee for Advanced Therapies. Draft reflection paper on classification of advanced-therapy medicinal products. EMA/CAT/600280/2010 Rev.1. (European Medicines Agency, 2015).

  100. 100.

    Centre for Biologics Evaluation and Research. Recommendations for Microbial Vectors use for Gene Therapy. U.S. Food & Drug Administration (2016). A useful set of guidelines describing the types of information necessary for regulatory approval of engineered bacteria as human therapeutics.

  101. 101.

    & The bioenergetic costs of a gene. Proc. Natl Acad. Sci. USA 112, 15690–15695 (2015).

  102. 102.

    , , & Quantifying cellular capacity identifies gene expression designs with reduced burden. Nat. Methods 12, 415–418 (2015).

  103. 103.

    , , , & In vivo gene expression dynamics of tumor-targeted bacteria. ACS Synth. Biol. 1, 465–470 (2012).

  104. 104.

    , , , & 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat. Chem. Biol. 12, 82–86 (2015).

  105. 105.

    , & Listeria monocytogenes-based antibiotic resistance gene-free antigen delivery system applicable to other bacterial vectors and DNA vaccines. Infect. Immun. 72, 6418–6425 (2004).

  106. 106.

    & Visualization of evolutionary stability dynamics and competitive fitness of Escherichia coli engineered with randomized multigene circuits. ACS Synth. Biol. 2, 519–528 (2013).

  107. 107.

    , , , & Minimal model of ribosome allocation dynamics captures trade-offs in expression between endogenous and synthetic genes. ACS Synth. Biol. 5, 710–720 (2016).

  108. 108.

    , , , & A 'resource allocator' for transcription based on a highly fragmented T7 RNA polymerase. Mol. Syst. Biol. 10, 742 (2014).

  109. 109.

    et al. Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli. N. Biotechnol. 33, 78–90 (2016).

  110. 110.

    et al. Burden-driven feedback control of gene expression. bioRxiv (2017).

  111. 111.

    , & Fermentation by gut microbiota cultured in a simulator of the human intestinal microbial ecosystem is improved by supplementing a soygerm powder. J. Nutr. 130, 2599–2606 (2000).

  112. 112.

    & Gut-on-a-Chip microenvironment induces human intestinal cells to undergo villus differentiation. Integr. Biol. 5, 1130 (2013).

  113. 113.

    et al. An intestinal organ culture system uncovers a role for the nervous system in microbe-immune crosstalk. Cell 168, 1135–1148.e12 (2017).

  114. 114.

    U.S. Securities and Exchange Commission. Partial clinical hold lifted and enrollment resumes for Aduro Biotech LADD clinical trials. SEC (2016).

  115. 115.

    et al. Genomically recoded organisms expand biological functions. Science 342, 357–360 (2013). A study describing removal of a codon from the entire genome of E. coli and repurposing it for the addition of non-native amino acids.

  116. 116.

    et al. Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA. Nucl. Acids Res. 45, 6971–6980 (2017).

  117. 117.

    et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature 518, 55–60 (2015).

  118. 118.

    et al. Defining synonymous codon compression schemes by genome recoding. Nature 539, 59–64 (2016).

  119. 119.

    et al. Recoded organisms engineered to depend on synthetic amino acids. Nature 518, 89–93 (2015).

  120. 120.

    et al. Design, synthesis, and testing toward a 57-codon genome. Science 353, 819–822 (2016).

  121. 121.

    et al. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat. Methods 10, 354–360 (2013). A landmark study describing the design and quantification of an extensive library of components to rationally modulate gene expression levels.

  122. 122.

    & Tunable protein degradation in bacteria. Nat. Biotechnol. 32, 1276–1281 (2014).

  123. 123.

    , , , & Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Bioeng. Bugs 3, 38–43 (2012).

  124. 124.

    et al. GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLOS ONE 6, e21622 (2011).

  125. 125.

    et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

  126. 126.

    et al. antiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences. Nucleic Acids Res. 39, W339–346 (2011).

  127. 127.

    et al. Massively parallel de novo protein design for targeted therapeutics. Nature 550, 74–79 (2017).

  128. 128.

    et al. Bacterial virulence phenotypes of Escherichia coli and host susceptibility determine risk for urinary tract infections. Sci. Transl Med. 9, eaaf1283 (2017).

  129. 129.

    , & Selection of bacterial virulence genes that are specifically induced in host tissues. Science 259, 686–688 (1993).

  130. 130.

    , , , & Identification of Lactobacillus plantarum genes that are induced in the gastrointestinal tract of mice. J. Bacteriol. 186, 5721–5729 (2004).

  131. 131.

    et al. The outer mucus layer hosts a distinct intestinal microbial niche. Nat. Commun. 6, 8292 (2015).

  132. 132.

    et al. A synthetic genetic edge detection program. Cell 137, 1272–1281 (2009).

  133. 133.

    , , & Engineering RGB color vision into Escherichia coli. Nat. Chem. Biol. 13, 706–708 (2017).

  134. 134.

    , , , & Modular, multi-input transcriptional logic gating with orthogonal LacI/GalR family chimeras. ACS Synth. Biol. 3, 645–651 (2014).

  135. 135.

    Disciplined approach to drug discovery and early development. Sci. Transl Med. 8, 349ps315 (2016). Perspective discussing an approach to drug discovery and development that hopes to increase the likelihood of success through discipline early in the conceptualization and testing of a potential therapy.

  136. 136.

    , , & Use of luciferase genes as biosensors to study bacterial physiology in the digestive tract. Appl. Environ. Microbiol. 64, 2721–2722 (1998).

  137. 137.

    , & Genetic marking of Lactococcus lactis shows its survival in the human gastrointestinal tract. Appl. Environ. Microbiol. 61, 2771–2774 (1995).

  138. 138.

    3rd & Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic. Infect. Immun. 55, 3035–3043 (1987).

  139. 139.

    , & Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res. 57, 4537–4544 (1997).

  140. 140.

    et al. Listeria-based cancer vaccines that segregate immunogenicity from toxicity. Proc. Natl Acad. Sci. USA 101, 13832–13837 (2004).

  141. 141.

    , , , & Construction and characterization of an attenuated Listeria monocytogenes strain for clinical use in cancer immunotherapy. Clin. Vaccine Immunol. 16, 96–103 (2009).

  142. 142.

    et al. Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice. Proc. Natl Acad. Sci. USA 108, 6252–6257 (2011).

  143. 143.

    et al. Mining the human gut microbiota for immunomodulatory organisms. Cell 168, 928–943.e11 (2017).

  144. 144.

    , , & Programming a human commensal bacterium, Bacteroides thetaiotaomicron, to sense and respond to stimuli in the murine gut microbiota. Cell Syst. 1, 62–71 (2015).

  145. 145.

    , & Tunable expression tools enable single-cell strain distinction in the gut microbiome. Cell 169, 538–546.e12 (2017).

  146. 146.

    , , & Engineered regulatory systems modulate gene expression of human commensals in the gut. Cell 169, 547–558.e15 (2017).

  147. 147.

    , & Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342 (2000).

  148. 148.

    et al. Synthetic gene networks that count. Science 324, 1199–1202 (2009).

  149. 149.

    & Synthetic biology. Genomically encoded analog memory with precise in vivo DNA writing in living cell populations. Science 346, 1256272 (2014).

  150. 150.

    et al. Permanent genetic memory with >1-byte capacity. Nat. Methods 11, 1261–1266 (2014).

  151. 151.

    , & Rewritable digital data storage in live cells via engineered control of recombination directionality. Proc. Natl Acad. Sci. USA 109, 8884–8889 (2012).

  152. 152.

    , , & Combinatorial synthesis of genetic networks. Science 296, 1466–1470 (2002).

  153. 153.

    et al. Complex cellular logic computation using ribocomputing devices. Nature 548, 117–121 (2017).

  154. 154.

    , , , & Synthetic recombinase-based state machines in living cells. Science 353, aad8559 (2016). Development of state machines using DNA recombinase recognition sites. Circuits based on this concept could be used to integrate both the presence and order of several signals for diagnosis or control of therapeutic expression in the future.

  155. 155.

    & A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000).

  156. 156.

    et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516–519 (2008).

  157. 157.

    , , & Synchronous long-term oscillations in a synthetic gene circuit. Nature 538, 514–517 (2016).

  158. 158.

    et al. Essential information for synthetic DNA sequences. Nat. Biotechnol. 29, 22; discussion 22–23 (2011). Details the importance of sharing full DNA sequence information about all published synthetic circuits.

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Acknowledgements

The authors thank A. Naydich for critical review of the manuscript. D.T.R. is supported by a Human Frontier Science Program Long-Term Fellowship and a National Health and Medical Research Council (NHMRC) RG Menzies Early Career Fellowship from the Menzies Foundation through the Australian NHMRC.

Author information

Affiliations

  1. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    • David T. Riglar
    •  & Pamela A. Silver
  2. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA.

    • David T. Riglar
    •  & Pamela A. Silver

Authors

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Contributions

D.T.R. researched data for the article and wrote the article. D.T.R. and P.A.S. substantially contributed to the discussion of content and reviewed and edited the manuscript before submission.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Pamela A. Silver.

Supplementary information

Word documents

  1. 1.

    Supplementary information S1 (table)

    Completed, Active and Proposed clinical trials involving engineered live bacteria as therapeutics.

  2. 2.

    Supplementary information S2 (table)

    Examples of pre-clinical studies testing therapeutic engineered bacteria in animal disease models.

Glossary

Prodrug

An inactive form of a drug that requires activation, often by enzymatic cleavage, before adopting its therapeutic form.

Toll-like receptor

(TLR). A class of membrane receptors used by the innate immune system to recognize microbial molecules.

Metastasis

The spread of cancer cells from the original tumour to secondary sites around the body.

Colitis

Inflammation of the colon.

Trefoil factors

(TFFs). A family of peptides that are expressed at mucous membranes, including the gastrointestinal mucosa, and may have a protective role.

Recombinases

Enzymes that catalyse the excision, insertion, inversion or translocation of DNA between sites of specific DNA sequence.

Memory circuit

A genetic circuit that is designed to encode an extended response, or memory, following a transient cellular event.

Burden

The combined resources required by a cell to operate a given synthetic genetic pathway.

Quorum sensing

A common mechanism by which bacteria naturally sense the local population density of their own or other bacterial species to enable density- dependent cellular responses. Bacteria produce and sense a specific quorum sensing molecule. Constant secretion ensures that concentrations only reach threshold levels and change downstream transcriptional profiles when many bacteria are present in the population.

Kill switches

Circuits used as safety mechanisms to prevent incorrect activity of an engineered bacterial strain, usually by attempting to kill it or to prevent engineered functions.

Probiotics

Live microorganisms that are beneficial to health.

Genetic firewalls

Changes to the underlying genetic code of an organism in an attempt to prevent the possibility of effective genetic exchange between the engineered strain and other bacteria in the environment.

Log reduction

Measure of reduced bacterial growth based on the logarithm (base 10). Every additional log reduction therefore corresponds to tenfold lower growth or survival.

Logic gates

Circuits that use Boolean logic to activate an output only when a given combination of inputs is present.

State machines

Devices able to exist in one ofseveral unique states depending on the history of its inputs.

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DOI

https://doi.org/10.1038/nrmicro.2017.172