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Developinga new class of engineered live bacterial therapeutics to treat human diseases

Developinga new class of engineered live bacterial therapeutics to treat human diseases PERSPECTIVE https://doi.org/10.1038/s41467-020-15508-1 OPEN Developing a new class of engineered live bacterial therapeutics to treat human diseases 1 1 1 1 Mark R. Charbonneau , Vincent M. Isabella , Ning Li & Caroline B. Kurtz A complex interplay of metabolic and immunological mechanisms underlies many diseases that represent a substantial unmet medical need. There is an increasing appreciation of the role microbes play in human health and disease, and evidence is accumulating that a new class of live biotherapeutics comprised of engineered microbes could address specific mechanisms of disease. Using the tools of synthetic biology, nonpathogenic bacteria can be designed to sense and respond to environmental signals in order to consume harmful compounds and deliver therapeutic effectors. In this perspective, we describe considerations for the design and development of engineered live biotherapeutics to achieve regulatory and patient acceptance. he human body is host to diverse microbial communities, and the complex interactions between the host and its microbial counterparts play a key role in human health and Tdisease . Notably, members of the microbial community inhabiting the human gastro- intestinal tract (termed the gut microbiota) contribute to several metabolic and immune- 2 3 4 mediated diseases, including obesity , malnutrition , intestinal inflammatory disease , as well as 5,6 to anti-cancer immunity . The discovery of these host–microbe interactions presents the opportunity to address disease by modulating the structure and function of the gut microbiota. The field of synthetic biology applies the principles of molecular biology and metabolic engi- neering to design biological circuits that can be applied to medicine. A wide array of tools has been developed for several microbial host organisms, or chassis, that enable investigators to engineer mechanisms to address disease . Engineered bacterial strains can be designed to sense and respond to environmental signals within the body, including those in the gastrointestinal 8,9 tract or in the microenvironment of solid tumors . In this Perspective, we describe the opportunities for and challenges facing the application of synthetic biology tools to the devel- opment of therapeutics for human disease. We explore regulatory considerations for the development of engineered live biotherapeutic organisms as medicines and discuss strategies for how these therapeutics can be evaluated for their pharmacokinetic and pharmacodynamic properties. Lastly, we address considerations for manufacturability of engineered microbes to enable production at scale, as well as formulations and presentations that support the needs of patients. Regulatory considerations for live biotherapeutic products Live biotherapeutic products (LBPs) are defined as live organisms designed and developed to treat, cure, or prevent a disease or condition in humans . Notably, LBPs exclude vaccines, filterable viruses, oncolytic viruses, and organisms used as vectors for transferring genes into the host. LBPs are distinguished from probiotic supplements on the basis of their labeling claims, as 1 ✉ Synlogic, Inc., 301 Binney Street, Cambridge, MA 02142, USA. email: caroline@synlogictx.com NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications 1 1234567890():,; PERSPECTIVE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 most probiotics are regulated as dietary supplements and cannot the organism to replicate or persist in the host and/or the 10–12 make claims to treat or prevent disease . However, some environment can also be characterized, and it may be beneficial to probiotics may fit the definition of LBPs and can be developed as incorporate biocontainment strategies to restrict replication of the such if they have potential efficacy with respect to disease. LBPs candidate strain within the body. (4) Regardless of the inclusion can include genetically modified organisms (recombinant LBPs) if of biocontainment strategies, the residence time and elimination they have been engineered by adding, deleting, or altering genetic of the engineered organism within the body should be deter- material within the organism . In both the United States and mined. One approach to better characterize the strain is to first Europe, development of LBPs requires the demonstration of study clearance of the orally administered chassis organism in quality by establishing safety, reliability, robustness, and con- feces of non-human primates and healthy volunteers . (5) Lastly, 10,13 sistency of each batch produced . They must also be studied in the biodistribution of the engineered organism outside its target well-controlled clinical trials in the intended patient population to site (e.g., the gastrointestinal tract or solid tumors) may be 10–12 establish safety and efficacy . important to determine. In the United States, recombinant LBPs are regulated by the Food and Drug Administration (FDA) through the Center for Biologics Evaluation and Research (CBER). While there have Design of engineered therapeutic strains for the human gut been numerous probiotics approved as nutritional supplements Many bacterial species have been evolutionarily selected for and some engineered bacterial strains have been studied in the metabolic function within the mammalian gastrointestinal tract, 14–16 clinic , the FDA has not approved a live biotherapeutic and some probiotic organisms have a long history of safe use in 18,19 product for medicinal use to date. In 2016, the FDA issued a humans . Engineered LBPs may be designed to sense and guidance document describing the regulatory considerations for respond to features of the gut environment and represent an conducting clinical trials with LBPs . During development of opportunity to influence host biology in situ. Engineered bacterial engineered bacterial strains for therapeutic applications, the therapeutics can also incorporate biocontainment strategies, such microorganism must be well characterized and must be evaluated as auxotrophies that limit bacterial replication in the absence of a in clinical trials conducted under an investigational new drug provided metabolite. More sophisticated approaches for engi- application (IND). Regular interaction with regulatory authorities neered biocontainment have been conceived , but have yet to be is beneficial, as there is minimal precedent in the field, and the deployed in therapeutic applications. In some instances, it is current regulatory guidance documents are very general. For preferable to limit bacterial residence and replication to promote example, the FDA does not provide specific recommendations predictable and reproducible pharmacologic properties of the based on the site of action or therapeutic indication the LBP is engineered therapeutic. A non-colonizing strain, coupled with a intended to treat. Each LBP will have unique properties, including mechanism of biocontainment, may be well suited to achieve colonization, clearance, microbial products, and delivery mod- this goal. alities (e.g., oral, topical, or injectable). These factors may result in Escherichia coli Nissle 1917 (EcN) has been used as a probiotic different requirements to demonstrate that the LBP is safe and since its isolation over 100 years ago . In its unengineered form, efficacious . Bacterial components of the chassis, such as lipo- EcN has been used to treat various gastrointestinal conditions, polysaccharides, are of less concern for an oral therapeutic but including inflammatory bowel disease and irritable bowel 18,19 may have significant ramifications for safety when delivered syndrome . EcN is believed to impede the growth of oppor- systemically or intratumorally. In some cases, minimal toxicology tunistic pathogens, including Salmonella spp. and other coliform studies may be needed, if the agent is not disseminated from a enteropathogens, through the production of microcin proteins or 18,22,23 local site. However, if there is a risk that the organism may reach production of iron-scavenging siderophores . Additionally, other tissues, additional studies could be required to support the EcN may interact with the intestinal epithelium to stimulate anti- safety of the LBP. Since there is no published guidance that inflammatory activities , as well as to restore and maintain outlines toxicology requirements for LBPs specifically, the path intestinal barrier function . Notably, EcN does not exhibit long- for development of a particular clinical candidate must be dis- term colonization in healthy humans after oral administration . cussed with the regulatory authorities in the region or country for This is likely due to ecological stability of the human gut the intended development and use of the product. microbiota and exclusion of incoming new bacteria through a To be approved for medicinal use, the facility in which the phenomenon termed colonization resistance . microorganism is manufactured, processed, and packaged should An additional advantage of EcN as a chassis organism for operate under regulations of current good manufacturing pro- engineered biotherapeutics is the wealth of knowledge about cesses (cGMP). The specific requirements for development of transcriptional and translational control of gene expression in engineered live bacterial therapeutics in the European Union strains of E. coli. This knowledge can be leveraged to engineer remain to be defined and may differ from those in the United EcN to respond to the environment within the human gastro- States. The European Pharmacopeia published a monograph intestinal tract. For example, several anaerobic-inducible pro- setting the quality standards for LBPs for human use, in European moters have been characterized in E. coli , which allow for Pharmacopoeia, Supplement 9.7; effective in April 2019 . induction of engineered circuits in the anoxic gut environment, Specific additional considerations concerning the clinical without undesired activation during production of biomass. In development of engineered bacterial therapeutics include the some instances, it may be preferable to activate effector functions following: (1) The genetic sequence of exogenously introduced under more specific conditions, rather than constitutively genes, including a high-quality, complete genome sequence for throughout the gastrointestinal tract. For example, coupling gene the engineered clinical candidate strain, may be provided to expression to biosensors of reactive oxygen and nitrogen species regulators, together with evidence supporting the stability of for the treatment of inflammatory bowel disease may help deliver 7,28 strain modifications over time. (2) It is highly preferable that the effectors specifically where activity is beneficial . Regulating engineered organism be unable to horizontally transfer antibiotic effector expression in response to bacterial quorum sensing 29 30 31–33 34 resistance cassettes to other members of the resident microbiota. molecules ,pH , specific carbon sources , temperature ,or One way to address this concern is to eliminate all known or combinations of these signals may allow for exquisitely tuned suspected antibiotic resistance genes used in the creation of the effector functions in various intestinal microenvironments . strain or present in the chassis microorganism. (3) The ability of Mining the extensive transcriptomic data available in E. coli can 2 NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 PERSPECTIVE Table 1 Engineered bacterial therapeutics currently in clinical development. Engineered bacterial Chassis organism Therapeutic indication Sponsor Phase of therapeutic development AG013 Lactococcus lactis Oral mucositis Oragenics Phase 2b AG014 Lactococcus lactis Gastrointestinal Inflammation in Primary ActoBio Therapeutics Phase 1 Immunodeficiency AG019 Lactococcus lactis Type 1 Diabetes Mellitus ActoBio Therapeutics Phase 1b/2a ADXS-HOT Lysteria monocytogenes Non-Small Cell Lung Cancer Advaxis Immunotherapies Phase 1 ADXS-HPV Lysteria monocytogenes HPV-Associated Cancers Advaxis Immunotherapies Phase 1/2 ADXS-PA Lysteria monocytogenes Metastatic Prostate Cancer Advaxis Immunotherapies Phase 2 APS001F Bifidobacterium longum Solid Tumors Anaeropharma Science Phase 1 AZT-04 Staphylococcus epidermidis Cancer Therapy-associated Rashes Azitra Phase 1 bacTRL-IL-12 Bifidobacterium longum Solid Tumors Symvivo Phase 1 SYNB1020 E. coli Nissle 1917 Hyperammonemia Synlogic Discontinued SYNB1618 E. coli Nissle 1917 Phenylketonuria (PKU) Synlogic Phase 1/2a SYNB1891 E. coli Nissle 1917 Solid Tumors Synlogic Phase 1 VXM01 Salmonella Typhi Ty21a Progressive Glioblastoma VAXIMM Phase 2 List of engineered bacterial therapeutics in clinical development, describing the chassis organism, therapeutic indication, and the organization sponsoring development. also provide information on endogenous promoters capable of strains in clinical development using the chassis organism Lac- sensing and responding to such signals, which can subsequently tococcus lactis (Table 1). 7,29,35 be engineered to regulate specific effector functions . With recent advances in molecular biology, some groups have Several genetically modified EcN strains have been developed turned their attention to bacterial chassis that were not previously as intestinal-acting antimicrobial agents and evaluated in pre- amenable to genetic manipulation. For example, CHAIN biotech clinical models. For example, Hwang et. al. demonstrated that the has developed a modified Clostridium strain capable of producing EcN-based gastrointestinal delivery of anti-biofilm enzyme, dis- the anti-inflammatory metabolite, β-hydroxybutyrate. This engi- persin B (DspB), resulted in a reduction of pre-colonized P. neered strain can be administered as spores that selectively ger- aeruginosa abundance in both nematode and murine models . minate in the colon to bypass key challenges associated with oral Other groups have reported the successful production of anti- delivery, including survival upon exposure to stomach acids, bile microbial peptides from EcN that are effective for significantly salts, and digestive enzymes. Other groups have focused on decreasing murine colonization by Enterococcal species or organisms that are able to colonize the gastrointestinal tract, 28 43 Salmonella typhimurium . In the face of increasingly prevalent including Bacteroides spp . This genus of bacteria is known for antibiotic-resistant pathogens, novel EcN-based antimicrobials harboring a diverse repertoire of enzymes for the breakdown 44–47 offer promise for the treatment of infections caused by organisms of host- and diet-derived carbohydrates . Recently, Shepherd recalcitrant to traditional approaches. In addition, engineered et al. engineered Bacteroides ovatus to metabolize porphyran, a bacterial therapeutics have the potential for increased specificity marine polysaccharide that is rarely encountered in a Western compared to broad-spectrum antibiotics, as these drugs may be diet . The resulting strain, B. ovatus NB001, was shown to stably tailored to target particular bacterial genera or species, and their engraft in the colonic microbiota of mice supplemented with function may be restricted to the gastrointestinal lumen. porphyran in the diet, and the fecal abundance of this strain was Other groups have constructed engineered EcN strains to treat titratable by modulating dietary porphyran . Novome Bio- metabolic disorders from within the gut. Chen et al. demon- technologies is developing this technology for clinical applica- strated that an N-acylphosphatidylethanolamine (NAPE)-pro- tions, and the use of such strains could be transformative for the ducing strain of EcN could significantly ameliorate symptoms treatment of chronic diseases. However, the genetic stability of an associated with high-fat diet feeding in mice. Mice treated with engineered live bacterial therapeutic is a concern for organisms this recombinant EcN displayed reduced adiposity, insulin that are intended to replicate within and/or colonize the patient’s resistance, and hepatosteatosis compared to animals treated with microbiota, and it may be possible for the strain to transfer an unengineered EcN control . Another group has engineered engineered genetic material to other members of the endogenous EcN to express genes responsible for the conversion of fructose, a microbiota. Gene cassettes conferring the ability to utilize por- prevalent sugar in the Western diet that contributes to metabolic phyran, for example, could be horizontally transferred to other disorders and cardiovascular disease, to mannitol, a prebiotic that members of the gut microbiota. While this genetic transfer is has been demonstrated to confer protection against metabolic unlikely to be directly harmful to the patient, it may eliminate the syndrome . competitive advantage of the engineered strain and undermine Probiotics in the genera Lactobacillus and Lactococcus have the efficacy of treatment. also attracted significant attention in the engineered biother- During selection of a bacterial chassis, as well as during apeutic arena. Though the genetic toolbox for these organisms is genetic circuit design, the complex biogeography of the gas- less advanced than that of E. coli, progress has been made with trointestinal tract can be considered for optimizing activity. regard to genetic modification and control of gene expression in Since the human colon is an anaerobic environment ,path- 39,40 these organisms . Similar to EcN, these genera do not colonize ways and enzymes that do not require oxygen are preferred. the human gut, thus allowing for predictive pharmacokinetic Moreover, while the colon harbors a diverse microbiota, these 41,42 profiling of therapeutic strains . These gram-positive organ- organisms are predominantly localized to a loosely adherent isms have evolved to survive in the harsh small intestinal envir- layer of mucus . Studies of radiolabeled E. coli in gnotobiotic onment, and the structure of their cell envelope is advantageous mice have demonstrated that mucus-adherent E. coli display for the secretion of effector proteins into the intestinal milieu. significantly higher rates of replication than those in the colonic Acto Bio Therapeutics currently has three engineered candidate lumen . Interestingly, no differences in replication rates were NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications 3 – Microenvironment for required function PERSPECTIVE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 – Effectors to be produced or consumed Circuit – Logic of gene regulation design – Selection of promoters and inducers Preferred Chassis strain – Biodistribution and selection characteristics elimination kinetics – Manufacturing feasibility – Optimal dose and dosage form Patient – Route of administration considerations – Biocontainment strategies Environment- Requirement sensing Solid for quantitative promoter oral dosing biomarkers system formulation Bio- Anaerobic Manufacturing and containment chassis clinical feasibility strategy organism Suitability for target environment Fig. 1 Considerations for the design of engineered live bacterial therapeutics. a Several aspects require consideration during the design of an engineered bacterial therapeutic. The selection of a chassis organism can be guided by the desired site of activity and pharmacokinetic properties of the chassis, as well as manufacturing feasibility. The design of genetic circuits may also be influenced by the circuit’s effectors, pragmatic concerns regarding inducer compounds, and the genetic stability of regulatory circuits. Critically, the design of an engineered bacterial drug may also be constrained by considerations for the needs of patients. b Optimal strain design often requires a balance between strain suitability for function in the target microenvironment and concerns for feasibility of manufacturing and clinical development. observed for Bacteroides thetaiotaomicron . Strict anaerobes, complicate manufacturing of these organisms. The chassis for a including Bacteroides spp., may be useful chassis organisms due bacterial therapeutic thus can be selected to meet both the to their abundance in the colonic microbiota of humans, as well requirements of its intended function and pragmatic con- as their capacity to consume complex dietary and host-derived siderations of translation to clinical application (Fig. 1a). These glycans . However, the need for strict anaerobiosis may selections in the design and development of engineered LBPs 4 NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 PERSPECTIVE frequently represent trade-offs, as optimization for manu- bacterial protein that induces apoptosis in tumor cells . This facturing or clinical feasibility may come at a cost to strain engineered strain suppressed the growth of tumors and prevented function in the body (Fig. 1b). For example, a solid oral dosing pulmonary metastasis in mice . Similarly, Li et al. engineered an formulation may be preferable foran LBP sincethisformat EcN chassis to produce cytotoxic compounds, including coli- could enable room temperature or refrigerated storage of the bactin, glidobactin, and luminmide to suppress tumor growth in a product in a patient’s home. However, the process of preparing mouse model . More recently, Ho et al. engineered EcN for the a lyophilized or spray-dried bacterial powder could result in treatment of colorectal cancer by expressing HlpA, a protein that 52,53 significant losses to cell viability and/or cell integrity . binds specifically to a heparan sulphate proteoglycan, enabling Similarly, incorporation of environmental sensors in the design engineered EcN to specifically target polyps in a murine colorectal of an engineered strain may enable exquisite control of engi- cancer model . The authors combined this polyp-targeting neered gene expression, but these sensors could also severely chassis with the secretion of myrosinase, an enzyme that con- constrain the acceptable parameters for processes used to pre- verts glucosinolates, a naturally occurring component of cruci- pare biomass. As such, the selection of a chassis organism, the ferous vegetables, to the chemopreventive metabolite, design of engineered gene circuits, and the development of sulphoraphane. The combination of HlpA and myrosinase manufacturing processes should be balanced to achieve the expression led to a significantly enhanced effect on tumor required characteristics of an LBP. regression and tumor occurrence in mice, compared to a construct Engineered bacterial therapeutics possess a potential advan- expressing myrosinase alone . In another report, Chowdhury tage over alternative microbiota-directed therapeutic approaches, et al. engineered a non-pathogenic E. coli strain to lyse specifically such as fecal microbiota transplants or defined consortia of within the tumor microenvironment and release an anti-CD47 naturally occurring species, in that genetic engineering can antagonist nanobody. The authors demonstrated the activation of confer functions that are not expressed by the endogenous tumor-infiltrating T cells, tumor regression, and long-term sur- microbiota. Engineered LBPs can be designed to perform natural vival in a syngeneic tumor model in mice, as well as abscopal biological processes, such as the assimilation of ammonia into effects on untreated tumors . amino acids, at significantly increased rates and to produce In addition to strains of E. coli, there is precedent for the use of effectors that are not native to bacteria, including human pro- anaerobic organisms for the treatment of cancer preclinically. For teins . Functions encoded by engineered bacteria also have example, strains of Bifidobacterium have been engineered to potential for the treatment of inborn errors of metabolism express cytosine deaminase (CD) in order to convert the relatively (IEMs) present in the host, such as phenylketonuria (PKU) . nontoxic compound 5-fluorocytosine (5-FC) into the cytotoxic Patients with PKU harbor genetic mutations that result in compound 5-fluorouracil (5-FU) in situ . Co-administration of a reduced activity of the enzyme, phenylalanine hydroxylase, CD-expressing Bifidobacterium infantis strain with 5-FC sig- which converts the essential amino acid phenylalanine (Phe) to nificantly inhibited tumor growth in mice . In a similar study, tyrosine. For PKU patients, dietary protein consumption elevates Wei et al. engineered Bifidobacterium longum to express the plasma Phe concentrations, and prolonged elevated plasma Phe proapoptotic compound, tumstatin . This strain was shown to can lead to severe cognitive impairment, among other sequelae. inhibit tumor growth in a mouse model by various routes of Synlogic has engineered a therapeutic strain of EcN, SYNB1618, administration . Given the plethora of preclinical data, engi- to degrade Phe by the expression of two distinct mechanisms: (1) neered variants of E. coli, Bifidobacterium, Salmonella, and Lis- the conversion of Phe to trans-cinnamic acid by the enzyme teria strains are all currently being evaluated clinically for the treatment of solid tumors (Table 1). phenylalanine ammonia lyase (PAL), and (2) the conversion of Phe to phenylpyruvic acid by the enzyme L-amino acid deami- Several aspects are critical for the design of engineered bacterial nase (LAAD) . Oral administration of SYNB1618 was shown to strains for treatment of tumors, including regulation of engi- significantly lower blood Phe concentrations in a mouse model neered circuits, selection of therapeutic effectors, safety and bio- of PKU, as well as to result in dose-dependent production of the containment within the tumor, and mode of delivery. Notably, PAL-specific urinary biomarker, hippuric acid, in healthy non- these aspects of strain design may interact and have significant human primates. A recent Phase 1/2a dose escalation study in implications for the translational potential of engineered live healthy volunteers and PKU patients that demonstrated that bacterial therapeutics. Chemically inducible promoters, including SYNB1618 was generally well tolerated (Clinicaltrials.gov Iden- the tetracycline inducible (Tet) promoter, are widely used in tifier: NCT03516487). This study also revealed a dose-dependent research applications to regulate engineered circuits, due to their production of hippuric acid upon administration of SYNB1618, ease of use and titratable expression. However, chemically indu- demonstrating Phe consumption by the engineered strain in cible promoters are less amenable to intratumoral applications, human subjects. since achieving an effective concentration of the inducer molecule in situ may be challenging, and some such compounds are not Generally Regarded As Safe (GRAS) for human use. Another Design of engineered therapeutic strains for solid tumors approach to regulation of engineered circuits is through quorum The notion of treating solid tumors with live bacteria was first sensing molecules, such as N-acyl-homoserine lactones (AHL), 55–57 69–73 reported more than 100 years ago . Solid tumors display that have been studied extensively in Salmonella strains .In abnormal blood vessel architecture, resulting in the development contrast to chemical induction, genetic circuits under the control of hypoxic regions and a necrotic core that can serve as suitable of oxygen sensitive promoters, such as the fumarate nitrate 35 27 habitats for obligate and facultative anaerobic bacteria. Preferential reductase (FNR) and the VHb promoter systems, could colonization of tumors upon administration in mice has been obviate the need for exogenously provided inducer compounds. demonstrated for a number of bacterial genera, including Bifido- Considering the heterogeneity of tumor architecture, other 58 59 60 9,61 bacterium , Clostridium , Salmonella ,and Escherichia . For environmental sensing systems, including temperature-inducible example, E. coli has been shown to colonize the region sur- promoters , may provide more consistent induction of engi- rounding the necrotic core of tumors after intravenous injection , neered circuits. However, temperature-inducible systems possess and several reports have demonstrated the use of engineered E. the significant drawback that circuit expression would not be 15,62–65 coli strains to treat solid tumors in preclinical models . limited to tumor tissue in the event of systemic strain Zhang et al. developed a strain of EcN to express azurin, a small dissemination. NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications 5 PERSPECTIVE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 One advantage of live bacterial therapeutics for treating cancer Testing strategies for engineered therapeutic organisms is that bacterial cells possess inherently proinflammatory prop- The low cost and high throughput of DNA synthesis and erties (e.g., TLR4 stimulation by bacterial lipopolysaccharides ). assembly, as well as bioinformatics tools to identify potential However, the selection of engineered therapeutic effectors is likely targets and effectors, together enable the rapid and cost-effective to be critical for efficacy in patients. While several examples of generation of prototype engineered strains . However, the effectors have shown promise preclinically, rigorous clinical trials requirements for clinical development of engineered biother- will be required to determine whether these results translate to apeutics, including diligent toxicology studies and adherence to heterogeneous human cancers that have been recalcitrant to regulatory guidelines, do not scale similarly and represent sig- current modes of therapy. nificant cost and effort for each candidate strain. Therefore, it will As a new platform for treating cancer patients, the safety of be advantageous to develop predictive testing strategies that can engineered live bacterial therapeutics is paramount, and bio- be applied in high throughput to characterize the function of containment strategies are recommended. An important con- engineered strains, to optimize potency, and to establish high sideration with respect to safety in patients that may have a confidence in translational potential prior to nomination of compromised immune system is that engineered bacterial pro- strains for clinical development. Figure 2 displays a schematic ducts are likely to engage both innate and adaptive immunity in representation of how this strategy for development of engineered the event of release of the organism into the body following bacterial biotherapeutics could be implemented. tumor lysis , triggering inflammatory responses. To help address Environmental conditions, including pH, oxygen concentra- these concerns, nutritional auxotrophies or kill switches can be tion, and nutrient availability, are major determinants of strain utilized in engineered strains to prevent replication inside the viability and metabolism, and the first line of testing for engi- host organism as well as to control the duration of therapeutic neered biotherapeutic organisms could utilize predictive, high activity and limit the potential toxicity of an engineered strain to throughput in vitro models that recapitulate the physiological patients. A simple “kill switch” strategy is to characterize the conditions of the target environment. For example, methods that antibiotic susceptibility of an engineered live bacterial therapeutic simulate the conditions of the human upper gastrointestinal tract, and to use these compounds in the event of suspected bacterial such as the simulated human intestinal microbial ecosystem dissemination from tumors. (SHIME), are useful for characterizing the viability and function Engineered bacterial therapeutic idea Prototype generation Rational pathway design Prototype strain construction In vitro simulations In vivo proof of mechanism Strain optimization High throughput enzyme screening Iterative optimization Expression and payload optimization Circuit troubleshooting (’Omics) Process development Pathway refactoring Incorporation of auxotrophies Lead selection Development of quantitative biomarkers Strain characterization - Organ-on-chip testing - Quantitative in silico modeling - In vivo disease models Manufacturability assessment Candidate selection Process scale-up Assay development - Potency and viability - In vivo biomarkers IND enabling studies - In vivo toxicology Engineered bacterial therapeutic candidate Fig. 2 Strategy for the development of engineered live bacterial therapeutic clinical candidates. Schematic representation of a workflow for developing clinical candidate-quality engineered strains. The development workflow should incorporate technologies for optimizing strain potency, as well as predictive in vitro and in vivo assays, as well quantitative pharmacology models, to maximize translational potential for patient populations. 6 NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 PERSPECTIVE 48,77 of engineered strains . These strains can be examined in iso- including small molecules and recombinant proteins, by model- lation, or in the context of diverse microbial communities that ing the pharmacokinetic and pharmacodynamic properties of represent the human gut microbiota. Moreover, these in vitro drug candidates across a wide assortment of therapeutic indica- simulations can elucidate the kinetics of engineered circuit reg- tions . These approaches will become increasingly important for ulation and function over timescales that are relevant to human the design and evaluation of engineered biotherapeutic organisms biology. for clinical development. A drawback to simplified in vitro simulations is the absence of human cells and tissue architecture. In recent years, significant Biomarkers of therapeutic activity advances have been made in organ-on-chip microfluidic systems The development of engineered therapeutic strains can be greatly that enable investigators to study the effects of engineered aided by incorporation of robust, quantitative biomarkers of microbes on various human tissues, including the permeability of strain function (e.g., metabolites or proteins produced by the microbial effectors across epithelial barriers and effects on tissue engineered strain directly) into strain design. These biomarkers viability . Recently, Jalili-Firoozinezhad et al. demonstrated the can elucidate the pharmacokinetics and/or pharmacodynamics of stable co-culture of human intestinal tissues that exhibited an the engineered strain and facilitate the translation from pre- intact mucus layer with a complex human gut microbiota under clinical models to clinical studies, enable proof of mechanism anaerobic conditions . Though this technology remains in its during early phase safety studies, and increase confidence in infancy, and compelling data sets that demonstrate its predictive predictions of efficacy for later phase clinical trials. Conversely, potential are needed to support its robust application to drug the absence of quantitative biomarkers can severely limit the development, organ-on-chip models represent an opportunity to information available to investigators about the function of an elucidate features of engineered strain function in physiologically engineered live bacterial therapeutic in humans prior to efficacy relevant environments early in the strain development process. studies in patients. An ideal biomarker for these purposes satisfies In vitro models of engineered strain function possess many four criteria: (1) the biomarker is mechanistically linked to the advantages, including high throughput and comparatively low designed function of the strain or to the disease itself, (2) the cost, but they are simplified representations of the host and its biomarker is quantifiable in noninvasively collected samples (e.g., associated microbiota. Therefore, animal models will remain a plasma, urine, or feces), (3) the biomarker has a quantitative critical component of testing strategies for engineered bacterial relationship to strain activity, and (4) the biomarker compound is therapeutics in the context of various diseases. The selection of an readily discernable from endogenous compounds in the sample appropriate animal model depends on the question being matrix (i.e., it is unique or produced at levels well above addressed, as the translational value of animal models varies by background). species and genotype. For example, while mice are readily avail- Quantitative biomarkers may not be available for all engineered able for preclinical studies, the oral bioavailability of small pathways, however, and the products of some engineered strains molecule drugs in humans and rodents have demonstrated poor may be unstable in host matrices or present at high concentra- correlation . Human gastrointestinal anatomy and physiology is tions endogenously. In some cases, it may be possible to identify more closely approximated by pigs and non-human primates metabolic conversions of microbial products that are performed than by rodent models , but models of disease states may be by host tissues. For example, the phenylalanine ammonia lyase unavailable in these large animal species. (PAL) enzyme expressed by the Phe consuming EcN strain, Animal models can be applied early in strain development to SYNB1618, produces trans-cinnamic acid, which is in turn con- evaluate performance characteristics of engineered prototypes. verted by host tissues into hippuric acid (HA) and excreted in the For example, rodent models can be used to obtain confidence that urine . Measurement of urinary HA provides a quantitative a prototype pathway is qualitatively active in vivo. Rodent models biomarker of strain activity that is directly linked to the strain’s are also suitable for determining whether heterologous gene intended function both in preclinical animal models as well as in expression results in a substantial fitness defect in an engineered safety studies with healthy volunteers. strain compared to the unmodified chassis organism. Such studies When a biomarker is not readily available from the design of can be conducted prior to deploying resources to optimize pro- an engineered pathway, in vivo pharmacology studies comparing totype pathway function (e.g., by screening homologous enzymes an engineered biotherapeutic strain to a negative control organ- or altering gene expression). However, it should be noted that ism lacking the therapeutic function, together with high high variability in animal models, together with relatively small throughput data acquisition methods, can be used for putative study sizes, may impair the statistical power of in vivo studies . biomarker identification . These methods could include both As such, quantitative studies of engineered biotherapeutic strain targeted and non-targeted metabolomics, proteomics, and high candidates in animals to demonstrate effects on disease states, as throughput RNA sequencing to identify transcriptional respon- well as comparative studies between prototypes, are most ses. Importantly, any potential biomarker requires rigorous appropriately considered only after strain characterization and experimental validation to determine whether it satisfies the cri- optimization in vitro. Importantly, these studies should consider teria listed above. the anticipated effect size, as well as variability in the model system, to ensure appropriate design. The function of engineered biotherapeutic strains in the host Manufacturability of engineered live bacterial therapeutics environment represents a complex, dynamic system. In the case Due to the unique characteristics of live bacteria, manufacturing of oral administration of an engineered organism, strain activity engineered bacterial therapeutics differs from other drug mod- is a function of gastric emptying, changing intestinal pH, oxygen alities in several respects, including development of the manu- and nutrient availability, strain viability, and dose. Predicting the facturing process, scale-up, and defining critical quality attributes translational potential of engineered bacterial therapeutics for the drug product. Considerable effort is warranted to develop necessitates a move toward mathematical frameworks for inte- robust fermentation and downstream processes to balance bio- grating data from in vitro and in vivo model systems to predict mass production and engineered circuit expression. In addition, the behavior of engineered strains in these dynamic conditions. predictive assays of strain activity are crucial to ensure the Quantitative systems pharmacology approaches have been widely potency of engineered bacterial therapeutics. For the purposes of used to accelerate drug development for other modalities, fermentation process development, bench scale bioreactors allow NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications 7 PERSPECTIVE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 for accurate measurement and control of pH, dissolved oxygen, patients, potentially leading to compliance issues and risks of temperature, as well as the automated addition of nutrients and product instability. Cold chain storage also presents a challenge chemical inducers during cell growth. In recent years, automated for supplying frozen drug products. Therefore, a solid formula- parallel bioreactor systems have accelerated fermentation process tion that is stable at room temperature is ideal for an orally development by combining smaller volumes and higher administered product. This requires that the live organism can throughput to enable greater iteration than traditional benchtop endure processes that convert a liquid culture to a solid form, 84–86 bioreactors . such as lyophilization or spray drying, to retain viability and Rational design of engineered bacterial therapeutics should potency. Technological advances including microencapsulation consider compatibility with manufacturing and clinical applica- and cryoprotectants could improve the stability of future LBP 90,91 tions. For example, residual concentrations of chemical inducers formulations , and buffering may be considered to preserve may be present after preparation of bacterial drug substance, cell activity and viability in the stomach. LBP formulations must necessitating additional purification steps if these inducers are also be palatable to patients to ensure compliance with dosing . used. In addition, not all chemical inducers have received GRAS For indications that require injection of the engineered live designation. For this reason, environmental sensors, including bacterial therapeutic, such as intratumoral administration, these oxygen and temperature sensitive regulators, are advantageous. drugs will be reconstituted and administered at a clinic that A key consideration for live bacterial products is cell viability specializes in this procedure, and frozen liquid formulations are during and after fermentation, downstream processing, for- feasible. Hydrogel formulations have also been used for delivery mulation, and storage. Traditionally, enumeration of LBPs has of intratumoral drugs and may improve the concentration of the relied on agar plating techniques to determine Colony Forming LBP within the tumor . For dermatological conditions, the LBP Units (CFU) , and while this methodology remains a staple for may be formulated as a cream or gel so that it can be applied the field, it may not be the most appropriate metric for all live topically by the patient, but engineered bacterial cells will require bacterial therapeutics. For example, cells in a viable but non- stability at the storage conditions needed for home use. Odor and culturable (VBNC) state may be unable to divide and form color masking may also be needed for any LBP to ensure patient colonies but may nonetheless retain sufficient metabolic activity compliance . to perform some engineered functions in situ . In this case, A quantitative biomarker of the strain’s activity in the body is assessment of viability using commercial live/dead stains to detect also very helpful to bridge early formulations with those used intact cell membranes may be more appropriate for enumerating later in development and commercialization. For example, a cells in the drug product . By contrast, the expression of mul- Phase 1 safety study could be conducted with a frozen liquid tistep metabolic pathways may require actively dividing bacterial preparation of cells to demonstrate activity of the LBP in humans, cells, making CFU plating more relevant. The ideal approach is while a solid oral formulation (e.g., a sachet or capsule) is being most appropriately determined for each engineered clinical can- developed. Production of the strain-specific biomarker can then didate strain. be used as a benchmark to bridge to solid formulations before The scale of manufacturing that is necessary for bacterial advancing into more lengthy and costly efficacy studies in therapeutics will be determined largely by dosing requirements, patients. and the efficacious dose of an engineered bacterial strain, in turn, is likely to be dependent on both its encoded mechanism of action and its route of administration. For example, metabolic conver- Limitations for developing engineered bacterial therapies sions in the gastrointestinal tract may require a larger dose of Several challenges and limitations to the development of live engineered cells than immunomodulatory mechanisms expressed engineered bacterial therapeutics are defined not by the tools of by intratumorally injected strains. This suggests that there will synthetic biology but rather by the lack of a clear mechanistic not be a “one size fits all” solution for the manufacture of engi- understanding of disease pathophysiology. A quantifiable rela- neered live bacterial therapeutics. tionship between the effectors expressed by an engineered An additional aspect that is unique to engineered bacterial drug organism and the underlying mechanisms of disease is necessary products is the need to ensure genetic stability during the pro- in order to engineer an optimal strain, but there remains a paucity duction of bacterial biomass. The inclusion of engineered genetic of disease- and effector-related biomarkers to establish dose- circuits encoding novel effector functions may come at a cost to response relationships, as well as target engagement at the site of bacterial fitness and/or growth rates, and this can lead to selective action. pressure for strain variants that have lost the engineered function Currently, non-colonizing engineered organisms require fre- and its associated fitness costs. To minimize this risk, engineered quent dosing, as the residence time of the strain is short in the components can be placed under tight regulatory control to body . However, it may be desirable to establish longer-term ensure that the engineered gene expression is stably maintained colonization by live bacterial therapeutics in the gastrointestinal in an “off” state until activation is desired during the preparation tract to allow for continuous delivery of a therapeutic effector, of biomass. Robust assays can also be implemented to ensure that thereby reducing the need for repeat dosing and enabling lower engineered circuit function is retained in the drug product. efficacious bacterial dose levels. This approach could also have the benefit of increasing patient receptivity and compliance, but biocontainment concerns would need to be addressed with reg- Considerations for dosing and formulations ulatory authorities. Understanding the ecological niche occupied Understanding the needs of the target patient population, as well by live bacterial therapeutics and how to enhance colonization in as practicality in clinical development, can be considered early the gut is an important area for continued investigation. during the development of an engineered live bacterial ther- Finally, expansion of the tools for synthetic biology, to allow apeutic. Bacterial cells grown in fermenters often must be purified commensal organisms to be considered as chassis for engineered and concentrated to yield a product that is suitable for dosing. For bacterial therapeutics, could enhance compatibility of the strain orally administered therapeutics, administration of a frozen sus- with the human host. Current bacterial synthetic biology tools are pension may be possible for in-clinic dosing (e.g., in Phase 1 most advanced for strains of E. coli and Lactobacillus, but there trials). However, for outpatient studies, formulations that require are comparatively few engineering tools for the diverse set of frozen storage and at-home reconstitution present challenges for strict anaerobes that reside in the colon. 8 NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 PERSPECTIVE Perspective and future developments 8. 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Developinga new class of engineered live bacterial therapeutics to treat human diseases

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PERSPECTIVE https://doi.org/10.1038/s41467-020-15508-1 OPEN Developing a new class of engineered live bacterial therapeutics to treat human diseases 1 1 1 1 Mark R. Charbonneau , Vincent M. Isabella , Ning Li & Caroline B. Kurtz A complex interplay of metabolic and immunological mechanisms underlies many diseases that represent a substantial unmet medical need. There is an increasing appreciation of the role microbes play in human health and disease, and evidence is accumulating that a new class of live biotherapeutics comprised of engineered microbes could address specific mechanisms of disease. Using the tools of synthetic biology, nonpathogenic bacteria can be designed to sense and respond to environmental signals in order to consume harmful compounds and deliver therapeutic effectors. In this perspective, we describe considerations for the design and development of engineered live biotherapeutics to achieve regulatory and patient acceptance. he human body is host to diverse microbial communities, and the complex interactions between the host and its microbial counterparts play a key role in human health and Tdisease . Notably, members of the microbial community inhabiting the human gastro- intestinal tract (termed the gut microbiota) contribute to several metabolic and immune- 2 3 4 mediated diseases, including obesity , malnutrition , intestinal inflammatory disease , as well as 5,6 to anti-cancer immunity . The discovery of these host–microbe interactions presents the opportunity to address disease by modulating the structure and function of the gut microbiota. The field of synthetic biology applies the principles of molecular biology and metabolic engi- neering to design biological circuits that can be applied to medicine. A wide array of tools has been developed for several microbial host organisms, or chassis, that enable investigators to engineer mechanisms to address disease . Engineered bacterial strains can be designed to sense and respond to environmental signals within the body, including those in the gastrointestinal 8,9 tract or in the microenvironment of solid tumors . In this Perspective, we describe the opportunities for and challenges facing the application of synthetic biology tools to the devel- opment of therapeutics for human disease. We explore regulatory considerations for the development of engineered live biotherapeutic organisms as medicines and discuss strategies for how these therapeutics can be evaluated for their pharmacokinetic and pharmacodynamic properties. Lastly, we address considerations for manufacturability of engineered microbes to enable production at scale, as well as formulations and presentations that support the needs of patients. Regulatory considerations for live biotherapeutic products Live biotherapeutic products (LBPs) are defined as live organisms designed and developed to treat, cure, or prevent a disease or condition in humans . Notably, LBPs exclude vaccines, filterable viruses, oncolytic viruses, and organisms used as vectors for transferring genes into the host. LBPs are distinguished from probiotic supplements on the basis of their labeling claims, as 1 ✉ Synlogic, Inc., 301 Binney Street, Cambridge, MA 02142, USA. email: caroline@synlogictx.com NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications 1 1234567890():,; PERSPECTIVE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 most probiotics are regulated as dietary supplements and cannot the organism to replicate or persist in the host and/or the 10–12 make claims to treat or prevent disease . However, some environment can also be characterized, and it may be beneficial to probiotics may fit the definition of LBPs and can be developed as incorporate biocontainment strategies to restrict replication of the such if they have potential efficacy with respect to disease. LBPs candidate strain within the body. (4) Regardless of the inclusion can include genetically modified organisms (recombinant LBPs) if of biocontainment strategies, the residence time and elimination they have been engineered by adding, deleting, or altering genetic of the engineered organism within the body should be deter- material within the organism . In both the United States and mined. One approach to better characterize the strain is to first Europe, development of LBPs requires the demonstration of study clearance of the orally administered chassis organism in quality by establishing safety, reliability, robustness, and con- feces of non-human primates and healthy volunteers . (5) Lastly, 10,13 sistency of each batch produced . They must also be studied in the biodistribution of the engineered organism outside its target well-controlled clinical trials in the intended patient population to site (e.g., the gastrointestinal tract or solid tumors) may be 10–12 establish safety and efficacy . important to determine. In the United States, recombinant LBPs are regulated by the Food and Drug Administration (FDA) through the Center for Biologics Evaluation and Research (CBER). While there have Design of engineered therapeutic strains for the human gut been numerous probiotics approved as nutritional supplements Many bacterial species have been evolutionarily selected for and some engineered bacterial strains have been studied in the metabolic function within the mammalian gastrointestinal tract, 14–16 clinic , the FDA has not approved a live biotherapeutic and some probiotic organisms have a long history of safe use in 18,19 product for medicinal use to date. In 2016, the FDA issued a humans . Engineered LBPs may be designed to sense and guidance document describing the regulatory considerations for respond to features of the gut environment and represent an conducting clinical trials with LBPs . During development of opportunity to influence host biology in situ. Engineered bacterial engineered bacterial strains for therapeutic applications, the therapeutics can also incorporate biocontainment strategies, such microorganism must be well characterized and must be evaluated as auxotrophies that limit bacterial replication in the absence of a in clinical trials conducted under an investigational new drug provided metabolite. More sophisticated approaches for engi- application (IND). Regular interaction with regulatory authorities neered biocontainment have been conceived , but have yet to be is beneficial, as there is minimal precedent in the field, and the deployed in therapeutic applications. In some instances, it is current regulatory guidance documents are very general. For preferable to limit bacterial residence and replication to promote example, the FDA does not provide specific recommendations predictable and reproducible pharmacologic properties of the based on the site of action or therapeutic indication the LBP is engineered therapeutic. A non-colonizing strain, coupled with a intended to treat. Each LBP will have unique properties, including mechanism of biocontainment, may be well suited to achieve colonization, clearance, microbial products, and delivery mod- this goal. alities (e.g., oral, topical, or injectable). These factors may result in Escherichia coli Nissle 1917 (EcN) has been used as a probiotic different requirements to demonstrate that the LBP is safe and since its isolation over 100 years ago . In its unengineered form, efficacious . Bacterial components of the chassis, such as lipo- EcN has been used to treat various gastrointestinal conditions, polysaccharides, are of less concern for an oral therapeutic but including inflammatory bowel disease and irritable bowel 18,19 may have significant ramifications for safety when delivered syndrome . EcN is believed to impede the growth of oppor- systemically or intratumorally. In some cases, minimal toxicology tunistic pathogens, including Salmonella spp. and other coliform studies may be needed, if the agent is not disseminated from a enteropathogens, through the production of microcin proteins or 18,22,23 local site. However, if there is a risk that the organism may reach production of iron-scavenging siderophores . Additionally, other tissues, additional studies could be required to support the EcN may interact with the intestinal epithelium to stimulate anti- safety of the LBP. Since there is no published guidance that inflammatory activities , as well as to restore and maintain outlines toxicology requirements for LBPs specifically, the path intestinal barrier function . Notably, EcN does not exhibit long- for development of a particular clinical candidate must be dis- term colonization in healthy humans after oral administration . cussed with the regulatory authorities in the region or country for This is likely due to ecological stability of the human gut the intended development and use of the product. microbiota and exclusion of incoming new bacteria through a To be approved for medicinal use, the facility in which the phenomenon termed colonization resistance . microorganism is manufactured, processed, and packaged should An additional advantage of EcN as a chassis organism for operate under regulations of current good manufacturing pro- engineered biotherapeutics is the wealth of knowledge about cesses (cGMP). The specific requirements for development of transcriptional and translational control of gene expression in engineered live bacterial therapeutics in the European Union strains of E. coli. This knowledge can be leveraged to engineer remain to be defined and may differ from those in the United EcN to respond to the environment within the human gastro- States. The European Pharmacopeia published a monograph intestinal tract. For example, several anaerobic-inducible pro- setting the quality standards for LBPs for human use, in European moters have been characterized in E. coli , which allow for Pharmacopoeia, Supplement 9.7; effective in April 2019 . induction of engineered circuits in the anoxic gut environment, Specific additional considerations concerning the clinical without undesired activation during production of biomass. In development of engineered bacterial therapeutics include the some instances, it may be preferable to activate effector functions following: (1) The genetic sequence of exogenously introduced under more specific conditions, rather than constitutively genes, including a high-quality, complete genome sequence for throughout the gastrointestinal tract. For example, coupling gene the engineered clinical candidate strain, may be provided to expression to biosensors of reactive oxygen and nitrogen species regulators, together with evidence supporting the stability of for the treatment of inflammatory bowel disease may help deliver 7,28 strain modifications over time. (2) It is highly preferable that the effectors specifically where activity is beneficial . Regulating engineered organism be unable to horizontally transfer antibiotic effector expression in response to bacterial quorum sensing 29 30 31–33 34 resistance cassettes to other members of the resident microbiota. molecules ,pH , specific carbon sources , temperature ,or One way to address this concern is to eliminate all known or combinations of these signals may allow for exquisitely tuned suspected antibiotic resistance genes used in the creation of the effector functions in various intestinal microenvironments . strain or present in the chassis microorganism. (3) The ability of Mining the extensive transcriptomic data available in E. coli can 2 NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 PERSPECTIVE Table 1 Engineered bacterial therapeutics currently in clinical development. Engineered bacterial Chassis organism Therapeutic indication Sponsor Phase of therapeutic development AG013 Lactococcus lactis Oral mucositis Oragenics Phase 2b AG014 Lactococcus lactis Gastrointestinal Inflammation in Primary ActoBio Therapeutics Phase 1 Immunodeficiency AG019 Lactococcus lactis Type 1 Diabetes Mellitus ActoBio Therapeutics Phase 1b/2a ADXS-HOT Lysteria monocytogenes Non-Small Cell Lung Cancer Advaxis Immunotherapies Phase 1 ADXS-HPV Lysteria monocytogenes HPV-Associated Cancers Advaxis Immunotherapies Phase 1/2 ADXS-PA Lysteria monocytogenes Metastatic Prostate Cancer Advaxis Immunotherapies Phase 2 APS001F Bifidobacterium longum Solid Tumors Anaeropharma Science Phase 1 AZT-04 Staphylococcus epidermidis Cancer Therapy-associated Rashes Azitra Phase 1 bacTRL-IL-12 Bifidobacterium longum Solid Tumors Symvivo Phase 1 SYNB1020 E. coli Nissle 1917 Hyperammonemia Synlogic Discontinued SYNB1618 E. coli Nissle 1917 Phenylketonuria (PKU) Synlogic Phase 1/2a SYNB1891 E. coli Nissle 1917 Solid Tumors Synlogic Phase 1 VXM01 Salmonella Typhi Ty21a Progressive Glioblastoma VAXIMM Phase 2 List of engineered bacterial therapeutics in clinical development, describing the chassis organism, therapeutic indication, and the organization sponsoring development. also provide information on endogenous promoters capable of strains in clinical development using the chassis organism Lac- sensing and responding to such signals, which can subsequently tococcus lactis (Table 1). 7,29,35 be engineered to regulate specific effector functions . With recent advances in molecular biology, some groups have Several genetically modified EcN strains have been developed turned their attention to bacterial chassis that were not previously as intestinal-acting antimicrobial agents and evaluated in pre- amenable to genetic manipulation. For example, CHAIN biotech clinical models. For example, Hwang et. al. demonstrated that the has developed a modified Clostridium strain capable of producing EcN-based gastrointestinal delivery of anti-biofilm enzyme, dis- the anti-inflammatory metabolite, β-hydroxybutyrate. This engi- persin B (DspB), resulted in a reduction of pre-colonized P. neered strain can be administered as spores that selectively ger- aeruginosa abundance in both nematode and murine models . minate in the colon to bypass key challenges associated with oral Other groups have reported the successful production of anti- delivery, including survival upon exposure to stomach acids, bile microbial peptides from EcN that are effective for significantly salts, and digestive enzymes. Other groups have focused on decreasing murine colonization by Enterococcal species or organisms that are able to colonize the gastrointestinal tract, 28 43 Salmonella typhimurium . In the face of increasingly prevalent including Bacteroides spp . This genus of bacteria is known for antibiotic-resistant pathogens, novel EcN-based antimicrobials harboring a diverse repertoire of enzymes for the breakdown 44–47 offer promise for the treatment of infections caused by organisms of host- and diet-derived carbohydrates . Recently, Shepherd recalcitrant to traditional approaches. In addition, engineered et al. engineered Bacteroides ovatus to metabolize porphyran, a bacterial therapeutics have the potential for increased specificity marine polysaccharide that is rarely encountered in a Western compared to broad-spectrum antibiotics, as these drugs may be diet . The resulting strain, B. ovatus NB001, was shown to stably tailored to target particular bacterial genera or species, and their engraft in the colonic microbiota of mice supplemented with function may be restricted to the gastrointestinal lumen. porphyran in the diet, and the fecal abundance of this strain was Other groups have constructed engineered EcN strains to treat titratable by modulating dietary porphyran . Novome Bio- metabolic disorders from within the gut. Chen et al. demon- technologies is developing this technology for clinical applica- strated that an N-acylphosphatidylethanolamine (NAPE)-pro- tions, and the use of such strains could be transformative for the ducing strain of EcN could significantly ameliorate symptoms treatment of chronic diseases. However, the genetic stability of an associated with high-fat diet feeding in mice. Mice treated with engineered live bacterial therapeutic is a concern for organisms this recombinant EcN displayed reduced adiposity, insulin that are intended to replicate within and/or colonize the patient’s resistance, and hepatosteatosis compared to animals treated with microbiota, and it may be possible for the strain to transfer an unengineered EcN control . Another group has engineered engineered genetic material to other members of the endogenous EcN to express genes responsible for the conversion of fructose, a microbiota. Gene cassettes conferring the ability to utilize por- prevalent sugar in the Western diet that contributes to metabolic phyran, for example, could be horizontally transferred to other disorders and cardiovascular disease, to mannitol, a prebiotic that members of the gut microbiota. While this genetic transfer is has been demonstrated to confer protection against metabolic unlikely to be directly harmful to the patient, it may eliminate the syndrome . competitive advantage of the engineered strain and undermine Probiotics in the genera Lactobacillus and Lactococcus have the efficacy of treatment. also attracted significant attention in the engineered biother- During selection of a bacterial chassis, as well as during apeutic arena. Though the genetic toolbox for these organisms is genetic circuit design, the complex biogeography of the gas- less advanced than that of E. coli, progress has been made with trointestinal tract can be considered for optimizing activity. regard to genetic modification and control of gene expression in Since the human colon is an anaerobic environment ,path- 39,40 these organisms . Similar to EcN, these genera do not colonize ways and enzymes that do not require oxygen are preferred. the human gut, thus allowing for predictive pharmacokinetic Moreover, while the colon harbors a diverse microbiota, these 41,42 profiling of therapeutic strains . These gram-positive organ- organisms are predominantly localized to a loosely adherent isms have evolved to survive in the harsh small intestinal envir- layer of mucus . Studies of radiolabeled E. coli in gnotobiotic onment, and the structure of their cell envelope is advantageous mice have demonstrated that mucus-adherent E. coli display for the secretion of effector proteins into the intestinal milieu. significantly higher rates of replication than those in the colonic Acto Bio Therapeutics currently has three engineered candidate lumen . Interestingly, no differences in replication rates were NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications 3 – Microenvironment for required function PERSPECTIVE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 – Effectors to be produced or consumed Circuit – Logic of gene regulation design – Selection of promoters and inducers Preferred Chassis strain – Biodistribution and selection characteristics elimination kinetics – Manufacturing feasibility – Optimal dose and dosage form Patient – Route of administration considerations – Biocontainment strategies Environment- Requirement sensing Solid for quantitative promoter oral dosing biomarkers system formulation Bio- Anaerobic Manufacturing and containment chassis clinical feasibility strategy organism Suitability for target environment Fig. 1 Considerations for the design of engineered live bacterial therapeutics. a Several aspects require consideration during the design of an engineered bacterial therapeutic. The selection of a chassis organism can be guided by the desired site of activity and pharmacokinetic properties of the chassis, as well as manufacturing feasibility. The design of genetic circuits may also be influenced by the circuit’s effectors, pragmatic concerns regarding inducer compounds, and the genetic stability of regulatory circuits. Critically, the design of an engineered bacterial drug may also be constrained by considerations for the needs of patients. b Optimal strain design often requires a balance between strain suitability for function in the target microenvironment and concerns for feasibility of manufacturing and clinical development. observed for Bacteroides thetaiotaomicron . Strict anaerobes, complicate manufacturing of these organisms. The chassis for a including Bacteroides spp., may be useful chassis organisms due bacterial therapeutic thus can be selected to meet both the to their abundance in the colonic microbiota of humans, as well requirements of its intended function and pragmatic con- as their capacity to consume complex dietary and host-derived siderations of translation to clinical application (Fig. 1a). These glycans . However, the need for strict anaerobiosis may selections in the design and development of engineered LBPs 4 NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 PERSPECTIVE frequently represent trade-offs, as optimization for manu- bacterial protein that induces apoptosis in tumor cells . This facturing or clinical feasibility may come at a cost to strain engineered strain suppressed the growth of tumors and prevented function in the body (Fig. 1b). For example, a solid oral dosing pulmonary metastasis in mice . Similarly, Li et al. engineered an formulation may be preferable foran LBP sincethisformat EcN chassis to produce cytotoxic compounds, including coli- could enable room temperature or refrigerated storage of the bactin, glidobactin, and luminmide to suppress tumor growth in a product in a patient’s home. However, the process of preparing mouse model . More recently, Ho et al. engineered EcN for the a lyophilized or spray-dried bacterial powder could result in treatment of colorectal cancer by expressing HlpA, a protein that 52,53 significant losses to cell viability and/or cell integrity . binds specifically to a heparan sulphate proteoglycan, enabling Similarly, incorporation of environmental sensors in the design engineered EcN to specifically target polyps in a murine colorectal of an engineered strain may enable exquisite control of engi- cancer model . The authors combined this polyp-targeting neered gene expression, but these sensors could also severely chassis with the secretion of myrosinase, an enzyme that con- constrain the acceptable parameters for processes used to pre- verts glucosinolates, a naturally occurring component of cruci- pare biomass. As such, the selection of a chassis organism, the ferous vegetables, to the chemopreventive metabolite, design of engineered gene circuits, and the development of sulphoraphane. The combination of HlpA and myrosinase manufacturing processes should be balanced to achieve the expression led to a significantly enhanced effect on tumor required characteristics of an LBP. regression and tumor occurrence in mice, compared to a construct Engineered bacterial therapeutics possess a potential advan- expressing myrosinase alone . In another report, Chowdhury tage over alternative microbiota-directed therapeutic approaches, et al. engineered a non-pathogenic E. coli strain to lyse specifically such as fecal microbiota transplants or defined consortia of within the tumor microenvironment and release an anti-CD47 naturally occurring species, in that genetic engineering can antagonist nanobody. The authors demonstrated the activation of confer functions that are not expressed by the endogenous tumor-infiltrating T cells, tumor regression, and long-term sur- microbiota. Engineered LBPs can be designed to perform natural vival in a syngeneic tumor model in mice, as well as abscopal biological processes, such as the assimilation of ammonia into effects on untreated tumors . amino acids, at significantly increased rates and to produce In addition to strains of E. coli, there is precedent for the use of effectors that are not native to bacteria, including human pro- anaerobic organisms for the treatment of cancer preclinically. For teins . Functions encoded by engineered bacteria also have example, strains of Bifidobacterium have been engineered to potential for the treatment of inborn errors of metabolism express cytosine deaminase (CD) in order to convert the relatively (IEMs) present in the host, such as phenylketonuria (PKU) . nontoxic compound 5-fluorocytosine (5-FC) into the cytotoxic Patients with PKU harbor genetic mutations that result in compound 5-fluorouracil (5-FU) in situ . Co-administration of a reduced activity of the enzyme, phenylalanine hydroxylase, CD-expressing Bifidobacterium infantis strain with 5-FC sig- which converts the essential amino acid phenylalanine (Phe) to nificantly inhibited tumor growth in mice . In a similar study, tyrosine. For PKU patients, dietary protein consumption elevates Wei et al. engineered Bifidobacterium longum to express the plasma Phe concentrations, and prolonged elevated plasma Phe proapoptotic compound, tumstatin . This strain was shown to can lead to severe cognitive impairment, among other sequelae. inhibit tumor growth in a mouse model by various routes of Synlogic has engineered a therapeutic strain of EcN, SYNB1618, administration . Given the plethora of preclinical data, engi- to degrade Phe by the expression of two distinct mechanisms: (1) neered variants of E. coli, Bifidobacterium, Salmonella, and Lis- the conversion of Phe to trans-cinnamic acid by the enzyme teria strains are all currently being evaluated clinically for the treatment of solid tumors (Table 1). phenylalanine ammonia lyase (PAL), and (2) the conversion of Phe to phenylpyruvic acid by the enzyme L-amino acid deami- Several aspects are critical for the design of engineered bacterial nase (LAAD) . Oral administration of SYNB1618 was shown to strains for treatment of tumors, including regulation of engi- significantly lower blood Phe concentrations in a mouse model neered circuits, selection of therapeutic effectors, safety and bio- of PKU, as well as to result in dose-dependent production of the containment within the tumor, and mode of delivery. Notably, PAL-specific urinary biomarker, hippuric acid, in healthy non- these aspects of strain design may interact and have significant human primates. A recent Phase 1/2a dose escalation study in implications for the translational potential of engineered live healthy volunteers and PKU patients that demonstrated that bacterial therapeutics. Chemically inducible promoters, including SYNB1618 was generally well tolerated (Clinicaltrials.gov Iden- the tetracycline inducible (Tet) promoter, are widely used in tifier: NCT03516487). This study also revealed a dose-dependent research applications to regulate engineered circuits, due to their production of hippuric acid upon administration of SYNB1618, ease of use and titratable expression. However, chemically indu- demonstrating Phe consumption by the engineered strain in cible promoters are less amenable to intratumoral applications, human subjects. since achieving an effective concentration of the inducer molecule in situ may be challenging, and some such compounds are not Generally Regarded As Safe (GRAS) for human use. Another Design of engineered therapeutic strains for solid tumors approach to regulation of engineered circuits is through quorum The notion of treating solid tumors with live bacteria was first sensing molecules, such as N-acyl-homoserine lactones (AHL), 55–57 69–73 reported more than 100 years ago . Solid tumors display that have been studied extensively in Salmonella strains .In abnormal blood vessel architecture, resulting in the development contrast to chemical induction, genetic circuits under the control of hypoxic regions and a necrotic core that can serve as suitable of oxygen sensitive promoters, such as the fumarate nitrate 35 27 habitats for obligate and facultative anaerobic bacteria. Preferential reductase (FNR) and the VHb promoter systems, could colonization of tumors upon administration in mice has been obviate the need for exogenously provided inducer compounds. demonstrated for a number of bacterial genera, including Bifido- Considering the heterogeneity of tumor architecture, other 58 59 60 9,61 bacterium , Clostridium , Salmonella ,and Escherichia . For environmental sensing systems, including temperature-inducible example, E. coli has been shown to colonize the region sur- promoters , may provide more consistent induction of engi- rounding the necrotic core of tumors after intravenous injection , neered circuits. However, temperature-inducible systems possess and several reports have demonstrated the use of engineered E. the significant drawback that circuit expression would not be 15,62–65 coli strains to treat solid tumors in preclinical models . limited to tumor tissue in the event of systemic strain Zhang et al. developed a strain of EcN to express azurin, a small dissemination. NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications 5 PERSPECTIVE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 One advantage of live bacterial therapeutics for treating cancer Testing strategies for engineered therapeutic organisms is that bacterial cells possess inherently proinflammatory prop- The low cost and high throughput of DNA synthesis and erties (e.g., TLR4 stimulation by bacterial lipopolysaccharides ). assembly, as well as bioinformatics tools to identify potential However, the selection of engineered therapeutic effectors is likely targets and effectors, together enable the rapid and cost-effective to be critical for efficacy in patients. While several examples of generation of prototype engineered strains . However, the effectors have shown promise preclinically, rigorous clinical trials requirements for clinical development of engineered biother- will be required to determine whether these results translate to apeutics, including diligent toxicology studies and adherence to heterogeneous human cancers that have been recalcitrant to regulatory guidelines, do not scale similarly and represent sig- current modes of therapy. nificant cost and effort for each candidate strain. Therefore, it will As a new platform for treating cancer patients, the safety of be advantageous to develop predictive testing strategies that can engineered live bacterial therapeutics is paramount, and bio- be applied in high throughput to characterize the function of containment strategies are recommended. An important con- engineered strains, to optimize potency, and to establish high sideration with respect to safety in patients that may have a confidence in translational potential prior to nomination of compromised immune system is that engineered bacterial pro- strains for clinical development. Figure 2 displays a schematic ducts are likely to engage both innate and adaptive immunity in representation of how this strategy for development of engineered the event of release of the organism into the body following bacterial biotherapeutics could be implemented. tumor lysis , triggering inflammatory responses. To help address Environmental conditions, including pH, oxygen concentra- these concerns, nutritional auxotrophies or kill switches can be tion, and nutrient availability, are major determinants of strain utilized in engineered strains to prevent replication inside the viability and metabolism, and the first line of testing for engi- host organism as well as to control the duration of therapeutic neered biotherapeutic organisms could utilize predictive, high activity and limit the potential toxicity of an engineered strain to throughput in vitro models that recapitulate the physiological patients. A simple “kill switch” strategy is to characterize the conditions of the target environment. For example, methods that antibiotic susceptibility of an engineered live bacterial therapeutic simulate the conditions of the human upper gastrointestinal tract, and to use these compounds in the event of suspected bacterial such as the simulated human intestinal microbial ecosystem dissemination from tumors. (SHIME), are useful for characterizing the viability and function Engineered bacterial therapeutic idea Prototype generation Rational pathway design Prototype strain construction In vitro simulations In vivo proof of mechanism Strain optimization High throughput enzyme screening Iterative optimization Expression and payload optimization Circuit troubleshooting (’Omics) Process development Pathway refactoring Incorporation of auxotrophies Lead selection Development of quantitative biomarkers Strain characterization - Organ-on-chip testing - Quantitative in silico modeling - In vivo disease models Manufacturability assessment Candidate selection Process scale-up Assay development - Potency and viability - In vivo biomarkers IND enabling studies - In vivo toxicology Engineered bacterial therapeutic candidate Fig. 2 Strategy for the development of engineered live bacterial therapeutic clinical candidates. Schematic representation of a workflow for developing clinical candidate-quality engineered strains. The development workflow should incorporate technologies for optimizing strain potency, as well as predictive in vitro and in vivo assays, as well quantitative pharmacology models, to maximize translational potential for patient populations. 6 NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 PERSPECTIVE 48,77 of engineered strains . These strains can be examined in iso- including small molecules and recombinant proteins, by model- lation, or in the context of diverse microbial communities that ing the pharmacokinetic and pharmacodynamic properties of represent the human gut microbiota. Moreover, these in vitro drug candidates across a wide assortment of therapeutic indica- simulations can elucidate the kinetics of engineered circuit reg- tions . These approaches will become increasingly important for ulation and function over timescales that are relevant to human the design and evaluation of engineered biotherapeutic organisms biology. for clinical development. A drawback to simplified in vitro simulations is the absence of human cells and tissue architecture. In recent years, significant Biomarkers of therapeutic activity advances have been made in organ-on-chip microfluidic systems The development of engineered therapeutic strains can be greatly that enable investigators to study the effects of engineered aided by incorporation of robust, quantitative biomarkers of microbes on various human tissues, including the permeability of strain function (e.g., metabolites or proteins produced by the microbial effectors across epithelial barriers and effects on tissue engineered strain directly) into strain design. These biomarkers viability . Recently, Jalili-Firoozinezhad et al. demonstrated the can elucidate the pharmacokinetics and/or pharmacodynamics of stable co-culture of human intestinal tissues that exhibited an the engineered strain and facilitate the translation from pre- intact mucus layer with a complex human gut microbiota under clinical models to clinical studies, enable proof of mechanism anaerobic conditions . Though this technology remains in its during early phase safety studies, and increase confidence in infancy, and compelling data sets that demonstrate its predictive predictions of efficacy for later phase clinical trials. Conversely, potential are needed to support its robust application to drug the absence of quantitative biomarkers can severely limit the development, organ-on-chip models represent an opportunity to information available to investigators about the function of an elucidate features of engineered strain function in physiologically engineered live bacterial therapeutic in humans prior to efficacy relevant environments early in the strain development process. studies in patients. An ideal biomarker for these purposes satisfies In vitro models of engineered strain function possess many four criteria: (1) the biomarker is mechanistically linked to the advantages, including high throughput and comparatively low designed function of the strain or to the disease itself, (2) the cost, but they are simplified representations of the host and its biomarker is quantifiable in noninvasively collected samples (e.g., associated microbiota. Therefore, animal models will remain a plasma, urine, or feces), (3) the biomarker has a quantitative critical component of testing strategies for engineered bacterial relationship to strain activity, and (4) the biomarker compound is therapeutics in the context of various diseases. The selection of an readily discernable from endogenous compounds in the sample appropriate animal model depends on the question being matrix (i.e., it is unique or produced at levels well above addressed, as the translational value of animal models varies by background). species and genotype. For example, while mice are readily avail- Quantitative biomarkers may not be available for all engineered able for preclinical studies, the oral bioavailability of small pathways, however, and the products of some engineered strains molecule drugs in humans and rodents have demonstrated poor may be unstable in host matrices or present at high concentra- correlation . Human gastrointestinal anatomy and physiology is tions endogenously. In some cases, it may be possible to identify more closely approximated by pigs and non-human primates metabolic conversions of microbial products that are performed than by rodent models , but models of disease states may be by host tissues. For example, the phenylalanine ammonia lyase unavailable in these large animal species. (PAL) enzyme expressed by the Phe consuming EcN strain, Animal models can be applied early in strain development to SYNB1618, produces trans-cinnamic acid, which is in turn con- evaluate performance characteristics of engineered prototypes. verted by host tissues into hippuric acid (HA) and excreted in the For example, rodent models can be used to obtain confidence that urine . Measurement of urinary HA provides a quantitative a prototype pathway is qualitatively active in vivo. Rodent models biomarker of strain activity that is directly linked to the strain’s are also suitable for determining whether heterologous gene intended function both in preclinical animal models as well as in expression results in a substantial fitness defect in an engineered safety studies with healthy volunteers. strain compared to the unmodified chassis organism. Such studies When a biomarker is not readily available from the design of can be conducted prior to deploying resources to optimize pro- an engineered pathway, in vivo pharmacology studies comparing totype pathway function (e.g., by screening homologous enzymes an engineered biotherapeutic strain to a negative control organ- or altering gene expression). However, it should be noted that ism lacking the therapeutic function, together with high high variability in animal models, together with relatively small throughput data acquisition methods, can be used for putative study sizes, may impair the statistical power of in vivo studies . biomarker identification . These methods could include both As such, quantitative studies of engineered biotherapeutic strain targeted and non-targeted metabolomics, proteomics, and high candidates in animals to demonstrate effects on disease states, as throughput RNA sequencing to identify transcriptional respon- well as comparative studies between prototypes, are most ses. Importantly, any potential biomarker requires rigorous appropriately considered only after strain characterization and experimental validation to determine whether it satisfies the cri- optimization in vitro. Importantly, these studies should consider teria listed above. the anticipated effect size, as well as variability in the model system, to ensure appropriate design. The function of engineered biotherapeutic strains in the host Manufacturability of engineered live bacterial therapeutics environment represents a complex, dynamic system. In the case Due to the unique characteristics of live bacteria, manufacturing of oral administration of an engineered organism, strain activity engineered bacterial therapeutics differs from other drug mod- is a function of gastric emptying, changing intestinal pH, oxygen alities in several respects, including development of the manu- and nutrient availability, strain viability, and dose. Predicting the facturing process, scale-up, and defining critical quality attributes translational potential of engineered bacterial therapeutics for the drug product. Considerable effort is warranted to develop necessitates a move toward mathematical frameworks for inte- robust fermentation and downstream processes to balance bio- grating data from in vitro and in vivo model systems to predict mass production and engineered circuit expression. In addition, the behavior of engineered strains in these dynamic conditions. predictive assays of strain activity are crucial to ensure the Quantitative systems pharmacology approaches have been widely potency of engineered bacterial therapeutics. For the purposes of used to accelerate drug development for other modalities, fermentation process development, bench scale bioreactors allow NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications 7 PERSPECTIVE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 for accurate measurement and control of pH, dissolved oxygen, patients, potentially leading to compliance issues and risks of temperature, as well as the automated addition of nutrients and product instability. Cold chain storage also presents a challenge chemical inducers during cell growth. In recent years, automated for supplying frozen drug products. Therefore, a solid formula- parallel bioreactor systems have accelerated fermentation process tion that is stable at room temperature is ideal for an orally development by combining smaller volumes and higher administered product. This requires that the live organism can throughput to enable greater iteration than traditional benchtop endure processes that convert a liquid culture to a solid form, 84–86 bioreactors . such as lyophilization or spray drying, to retain viability and Rational design of engineered bacterial therapeutics should potency. Technological advances including microencapsulation consider compatibility with manufacturing and clinical applica- and cryoprotectants could improve the stability of future LBP 90,91 tions. For example, residual concentrations of chemical inducers formulations , and buffering may be considered to preserve may be present after preparation of bacterial drug substance, cell activity and viability in the stomach. LBP formulations must necessitating additional purification steps if these inducers are also be palatable to patients to ensure compliance with dosing . used. In addition, not all chemical inducers have received GRAS For indications that require injection of the engineered live designation. For this reason, environmental sensors, including bacterial therapeutic, such as intratumoral administration, these oxygen and temperature sensitive regulators, are advantageous. drugs will be reconstituted and administered at a clinic that A key consideration for live bacterial products is cell viability specializes in this procedure, and frozen liquid formulations are during and after fermentation, downstream processing, for- feasible. Hydrogel formulations have also been used for delivery mulation, and storage. Traditionally, enumeration of LBPs has of intratumoral drugs and may improve the concentration of the relied on agar plating techniques to determine Colony Forming LBP within the tumor . For dermatological conditions, the LBP Units (CFU) , and while this methodology remains a staple for may be formulated as a cream or gel so that it can be applied the field, it may not be the most appropriate metric for all live topically by the patient, but engineered bacterial cells will require bacterial therapeutics. For example, cells in a viable but non- stability at the storage conditions needed for home use. Odor and culturable (VBNC) state may be unable to divide and form color masking may also be needed for any LBP to ensure patient colonies but may nonetheless retain sufficient metabolic activity compliance . to perform some engineered functions in situ . In this case, A quantitative biomarker of the strain’s activity in the body is assessment of viability using commercial live/dead stains to detect also very helpful to bridge early formulations with those used intact cell membranes may be more appropriate for enumerating later in development and commercialization. For example, a cells in the drug product . By contrast, the expression of mul- Phase 1 safety study could be conducted with a frozen liquid tistep metabolic pathways may require actively dividing bacterial preparation of cells to demonstrate activity of the LBP in humans, cells, making CFU plating more relevant. The ideal approach is while a solid oral formulation (e.g., a sachet or capsule) is being most appropriately determined for each engineered clinical can- developed. Production of the strain-specific biomarker can then didate strain. be used as a benchmark to bridge to solid formulations before The scale of manufacturing that is necessary for bacterial advancing into more lengthy and costly efficacy studies in therapeutics will be determined largely by dosing requirements, patients. and the efficacious dose of an engineered bacterial strain, in turn, is likely to be dependent on both its encoded mechanism of action and its route of administration. For example, metabolic conver- Limitations for developing engineered bacterial therapies sions in the gastrointestinal tract may require a larger dose of Several challenges and limitations to the development of live engineered cells than immunomodulatory mechanisms expressed engineered bacterial therapeutics are defined not by the tools of by intratumorally injected strains. This suggests that there will synthetic biology but rather by the lack of a clear mechanistic not be a “one size fits all” solution for the manufacture of engi- understanding of disease pathophysiology. A quantifiable rela- neered live bacterial therapeutics. tionship between the effectors expressed by an engineered An additional aspect that is unique to engineered bacterial drug organism and the underlying mechanisms of disease is necessary products is the need to ensure genetic stability during the pro- in order to engineer an optimal strain, but there remains a paucity duction of bacterial biomass. The inclusion of engineered genetic of disease- and effector-related biomarkers to establish dose- circuits encoding novel effector functions may come at a cost to response relationships, as well as target engagement at the site of bacterial fitness and/or growth rates, and this can lead to selective action. pressure for strain variants that have lost the engineered function Currently, non-colonizing engineered organisms require fre- and its associated fitness costs. To minimize this risk, engineered quent dosing, as the residence time of the strain is short in the components can be placed under tight regulatory control to body . However, it may be desirable to establish longer-term ensure that the engineered gene expression is stably maintained colonization by live bacterial therapeutics in the gastrointestinal in an “off” state until activation is desired during the preparation tract to allow for continuous delivery of a therapeutic effector, of biomass. Robust assays can also be implemented to ensure that thereby reducing the need for repeat dosing and enabling lower engineered circuit function is retained in the drug product. efficacious bacterial dose levels. This approach could also have the benefit of increasing patient receptivity and compliance, but biocontainment concerns would need to be addressed with reg- Considerations for dosing and formulations ulatory authorities. Understanding the ecological niche occupied Understanding the needs of the target patient population, as well by live bacterial therapeutics and how to enhance colonization in as practicality in clinical development, can be considered early the gut is an important area for continued investigation. during the development of an engineered live bacterial ther- Finally, expansion of the tools for synthetic biology, to allow apeutic. Bacterial cells grown in fermenters often must be purified commensal organisms to be considered as chassis for engineered and concentrated to yield a product that is suitable for dosing. For bacterial therapeutics, could enhance compatibility of the strain orally administered therapeutics, administration of a frozen sus- with the human host. Current bacterial synthetic biology tools are pension may be possible for in-clinic dosing (e.g., in Phase 1 most advanced for strains of E. coli and Lactobacillus, but there trials). However, for outpatient studies, formulations that require are comparatively few engineering tools for the diverse set of frozen storage and at-home reconstitution present challenges for strict anaerobes that reside in the colon. 8 NATURE COMMUNICATIONS | (2020) 11:1738 | https://doi.org/10.1038/s41467-020-15508-1 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15508-1 PERSPECTIVE Perspective and future developments 8. 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