- Email: [email protected]
Designer Probiotics: Paving the Way to Living Therapeutics
were always in a state of perpetual change and self-overcoming. It depends on us to use any new technology for our benefit, establishing and adopting rules and behaviors under ethical and moral terms. In this way, it will not be ‘appallingly obvious that our technology has exceeded our humanity’ as it was through the eyes of Albert Einstein (1879–1955). 1 Institute of History of Medicine and Public Health, Medical School, University of Lausanne, Lausanne, Switzerland
inexorable rise of antibiotic-resistant pathogens and the rather slow development of new antibiotics. Probiotics that deliver novel therapeutics efficiently and with site specificity are emerging living therapeutics that may transform existing paradigms of disease diagnosis and prevention.
Kluyveromyces lactis, and Pichia pastoris) are some of the prospective probiotics used for expressing heterologous genes encoding antimicrobial and anti-inflammatory biomolecules.
Designer Probiotics against Infectious Diseases As the efficacy of orally administered antigens is reduced during passage through the alimentary tract, probiotic-mediated drug delivery could be a promising strategy for administering multiple therapeutics (cytokines, antibody fragments, antigens, peptides, etc.) in situ at the site of infection, therefore circumventing the side effects associated with the systemic administration of drugs.
The normal microbiota are essential determinants of vital processes such as hematopoiesis, aging, immunity against infectious diseases, and behavior. Whereas the normal microbiota occa*Correspondence: [email protected] (.G. Papaioannou). sionally fails to protect the host against http://dx.doi.org/10.1016/j.tibtech.2017.04.011 pathogens, a perturbed gut microbiota is associated with inflammation, obesity, References 1. Karaberopoulos, D. et al. (2012) The theriac in antiquity. insulin resistance, diabetes, cardiovascu- For example, the vaccination of mice with Lancet 379, 1942–1943 lar diseases (CVDs), and neuropsychiatric recombinant Lactobacillus gasseri 2. Marx, V. (2015) Tissue engineering: organs from the lab. Nature 522, 373–377 disorders. NM713 expressing streptococcal M6 3. Aflalo, T. et al. (2015) Neurophysiology. Decoding motor protein (CRR6) protected them against imagery from the posterior parietal cortex of a tetraplegic The WHO has recently released a list of 12 streptococcus group A infections [5], human. Science 348, 906–910 4. Travis, J. (2015) Making the cut. Science 350, 1456–1457 antibiotic-resistant families of pathogenic and recombinant Lactococcus lactis 5. Brody, H. (2012) From an ethics of rationing to an ethics of bacteria, described as ‘priority patho- (LL-Thy 12) expressing human interleuwaste avoidance. N. Engl. J. Med. 366, 1949–1951 gens’, which pose a grave threat. Para- kin-10 (IL-10) provided relief from Crohn’s 6. Huxley, J. (1957) New Bottles for New Wine, Chatto & Windus doxically, the development of new disease (CD) [1]. Furthermore, L. lactis 7. Pepperell, R. (2005) Posthumans and extended experi- antibiotics has also slowed down, which strains that produce native (and pilinence. J. Evol. Technol. 14, 27–41 increases the demand for alternative ther- deleted) immunomodulatory surface pili8. Wehner, M.R. et al. (2014) Twitter: an opportunity for public health campaigns. Lancet 384, 131–132 apeutics. Expanding the efficacy of pro- ation appendages (SpaCBA) were found 9. Nabavi, S. et al. (2014) Engineering a memory with LTD biotics by introducing new genetic circuits to activate Toll-like receptor 2-dependent and LTP. Nature 511, 348–352 to deliver drug biomolecules is crucial. signaling in cell lines and to modulate 10. Wheeler, M.A. et al. (2016) Genetically targeted magnetic control of the nervous system. Nat. Neurosci. 19, 756–761 These recombinant probiotics, informally synthesis of anti-inflammatory cytokines 11. Min, B.K. and Muller, K.R. (2014) Electroencephalography/ called ‘designer probiotics’ or ‘probiotics (TNF-a, IL-6, IL-10, and IL-12) in human sonication-mediated human brain-brain interfacing tech2.0’, are poised to reduce the gap dendritic cells [6]. Another study reported nology. Trends Biotechnol. 32, 345–346 between the mounting antibiotic resis- recombinant L. lactis delivering therapeutance and the dearth of new antibiotics tic proteins at mucosal surfaces in murine [1–4]. models of human inflammatory bowel disScience & Society eases (IBDs) and human papillomavirus The science of probiotics, which was for- type 16 (HPV-16) [7]. merly confined to basic microbiology and food processing, has emerged in the In addition, epidemiological, experimenpostgenomic era of medicine and biology tal, and clinical evidence convincingly as a premier area of research towards shows that genitourinary microbiota domfunctional nutraceuticals, gastroenterol- inated by LAB protect the host against Birbal Singh,1,* Gorakh Mal,1 ogy, allergology, skin care, oncotherapy, bacterial vaginosis (BV) and sexually and Francesco Marotta2 psychoneuroendocrinology, and veteri- transmitted viral infections [9]. One connary applications. The lactic acid bacteria tributor to host protection is a group of Enhancing the functional reper- (LAB), bifidobacteria, Escherichia coli Nis- antimicrobial proteins including ribosotoire of probiotics is a promising sle 1917, and yeasts (Saccharomyces mally-synthesized bacteriocins, microapproach to cope with the cerevisiae, Saccharomyces boulardii, cins, peptides, and the type VI secretion 2
Biomedical Engineering Unit, First Department of Cardiology, Hippokration Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
Designer Probiotics: Paving the Way to Living Therapeutics
Trends in Biotechnology, August 2017, Vol. 35, No. 8
679
system (T6SS) of the gut and genitourinary and nasal tracts. These proteins are active at nanomolar levels and help protect against pathogens [8]. Langenaur and colleagues engineered vaginal-origin probiotic L. lactis and Lactobacillus jensenii to express HIV-inhibiting proteins for use as topical live microbiocides against HIV/AIDS and BV [9,10]. In addition to bacterial and viral infections, protozoan infections represent major health threats. The evidence that human gut microbiota elicit a protective immune response against Plasmodium spp. has led to the concept of evolving recombinant probiotics to cripple vector-borne diseases such as malaria, cholera, leishmaniasis, and trypanosomiasis. However, reports are currently scarce on recombinant probiotics against parasitic protozoa.
Anticancer Therapies Using Designer Probiotics The major challenge in oncology is a lack of availability of therapies against tumors with minimal toxicity to healthy tissues. Some approaches to treating cancer include chemotherapy, and prodrug delivery, targeted ultrasound, photodynamic therapy, biological therapy, and combinations of these strategies. Antitumor agents alone or in combina-
tion with radiotherapy or chemotherapy are toxic at therapeutic dosages. While reducing their dosage may lessen toxicity, their efficacy is also compromised. Certain bacteria, administered intravenously, colonize the hypoxic vicinities of tumors. In view of the ability of Bifidobacterium spp. and Clostridium spp. to preferentially proliferate in solid tumors, and currently aided by synthetic biology and genome engineering, there is renewed interest in using them as tumor-targeting vectors. Prodrug delivery methods, such as directed enzyme prodrug therapy (DEPT), use bacteria to express drug-converting enzymes (e.g., cytosine aminodehydrolase and nitroreductase). This approach may decrease the toxicity of drugs used in chemotherapy and eventually sensitize tumor cells to irradiation. Probiotics tailored to synthesize tumor-targeting peptides, induce cytotoxicity in tumor cells, or deliver chemotherapeutic drugs to remote tissues could be promising anticancer therapeutics.
colon carcinoma cells [11]. Pancreatitisassociated protein-I, expressed in a foodgrade recombinant L. lactis NZ9000, prevented experimentally induced intestinal mucositis in mouse models [12]. A proprietary oral rinse containing L. lactis, producing human trefoil factor 1 (hTFF1), ameliorated the severity of oral mucositis (OM) in a hamster model. The strategy is believed to be an effective tool for treating oral sores that develop following induction chemotherapy in patients suffering from head and esophageal cancer (Table 1). The programmable E. coli Nissle 1917, when administered orally, could detect hepatic metastasis signals in the urine of patients and is regarded as a live diagnostic probiotic [13].
Designer Probiotics and Metabolic Diseases
Endemic chronic diseases such as Alzheimer’s disease, autism, attention-deficit hyperactivity disorder, osteoarthritis, and stroke are increasing rapidly among human populations. Manipulating the gut ecosystem by recombinant probiotics expressing therapeutic biomolecules, fecal microbiota transplant, or phage Bioengineered L. lactis NZ9000-401- therapy might serve as alternative kiss1, producing KiSS1, a cancer-sup- approaches against lifestyle diseases. pressing peptide, is envisioned as a plausible anticancer probiotic by hampering When used as dietary supplements or migration and proliferation of human applied topically, designer probiotics
Table 1. Summary of Designer Probiotic and Postbiotic Formulations As Alternative Therapeutics Formulation (live component)
Manufacturer
Proposed applications
Actobiotics (L. lactis)
Intrexon, VA, USA
Targeted therapies against oral, gastrointestinal tract, metabolic, allergic, and autoimmune diseases AG013 attenuates OM and AG014 treats IBD through localized in situ delivery of anti-TNF-a Fab
AG013 (L. lactis secreting hTFF1)
ActoGenix N.V., Ghent, Belgium
Developing anti-TNF and bacterial formulation to prevent OM
CBM588 (Clostridium butyricum)
Osel Inc., CA, USA
Prevention of C. difficile infection and diarrheal disorders, restoring gut health after antibiotic therapy
LACTIN-V (Lactobacillus sp.)
Osel Inc., CA, USA
Restoring feminine genitourinary health by suppressing BV and viral infections
L. lactis
Oragenics, Tampa, FL, USA
Novel antibiotics, and proprietary probiotics for oral health of humans and pets
Multiple microbes
Seres Therapeutics, Cambridge, MA, USA
ActoBiotics drugs for treating diseases associated with dysbiosis
SYNB1020 (E. coli Nissle 1917)
Synlogic, Cambridge, MA, USA
Treating genetic metabolic diseases, IBDs including CD and ulcerative colitis, cardiovascular diseases, cancer, urea cycle disorders and hepatic encephalopathy (US Patent #9 487 464).
680
Trends in Biotechnology, August 2017, Vol. 35, No. 8
could support normal physiology and immunity to protect the host from infections, oxidative stress, inflammatory diseases, and autoimmune responses. For example, formulations that contain engineered probiotics producing human proinsulin and anti-inflammatory cytokines would provide a way to overcome the menace of diabetes. Orally administered L. lactis producing recombinant protein HSP65-6IA2P2 was found to prevent hyperglycemia, reduce insulitis, and increase glucose tolerance and regulatory
immune reactions in type 1 diabetes mellitus in nonobese diabetic mice [14]. Similarly, orally administered E. coli Nissle 1917 expressing N-acylphosphatidylethanolamines suppressed obesity in mice by regulating food intake. Because of similarities in eating behavior of mice and humans, the strategy might do well in humans [15].
Despite inconsistent and counterintuitive recognition for recombinant microbes and their metabolites, designer probiotics are envisaged as emerging living therapeutics for human and veterinary applicaConclusions and Perspectives Metabolic diseases and the surge of anti- tions. A combined therapy comprising an biotic resistance in pathogens are antimicrobial, anti-inflammatory, and
Ingeson/oral administraon
M Gut lumen
N
Designer probiocs
M D
Intravenous administraon (lyophilized bacteria)
N Urogenital healthcare:
Designer probiocs and peobioc matabolites
Restoring normal microflora, prevenon of allergy, an-HIV effects, protecon against BV/dysbiosis
Antumor acvies:
Ancancer effects:
Safe and selecve delivery of synthec genes to tumors; sensizing tumor cells to irradiaon
Producon of AMPs, CLAs, SCFAs, equol; removal of carcinogens; genotoxic matabolites
Anmicrobial effects:
DEPT/ CDEPT
Inhibion of angiogenesis, bacterial-mediated conversion of prodrugs to drugs in tumors
Food preservaon; inhibion of hospital-acquired infecons, IBD and parasic deseases, e.g., malaria
Disease diagnosis: Detecon of matastasis markers in body fluids and excreons
Tablets/ capsules
D
Gut epithelium Producon of proapoptoc or anproliferave factors (IL-10, TGF-β, SOD, catalase, SCFAs
serious public health concerns that require cost-effective inventive prescriptions. Microbes offer intriguing prospects for preventing illnesses that conventional medicines have failed to cure.
Managing obesity, depression, and cognive health
Sensing pathological/disease signals within gut; inhibiton of promitogenic and angiogenic acvies in cancer cells
Figure 1. A Diagrammatic Illustration of Health Benefits of Designer Probiotics (D) Instilled to Colonize the Mucosal Tissue. Synthetic biology and bioengineering are key research platforms to facilitate design and organization of the cell genome, create automated genetic circuits, and assemble large operons with integrated transcriptional and translational regulators. Designer probiotics are poised to benefit the host by eliminating pathogens, producing therapeutics (M) such as short chain fatty acids (SCFAs), antimicrobial peptides (AMPs), bacteriocins, enzyme inhibitors, cytokines, transforming growth factor-b (TGF-b), psychoactive compounds, conjugated linoleic acids (CLAs), and superoxide dismutase (SOD). Abbreviations: CDEPT, Clostridium directed enzyme prodrug therapy; DEPT, directed enzyme prodrug therapy; IBD, inflammatory bowel disease; N, normal microbiota.
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immunomodulatory repertoire of designer probiotics could combat infectious and metabolic diseases. Hence, probiotics that readily integrate with clinical prescriptions should be a prioritized area of research. Colonization of mucosal tissue could be a challenge for designer probiotics used as ‘drugs’, because of the intrinsic property of resident microbiota to resist instilled or invading microorganisms. Using humanorigin, commensal bacteria to deliver therapeutics could possibly resolve the problem. The safety of the end users and containment of exogenous genetic elements in recombinant probiotics are of concern. The likely probiotic-mediated rise of immune responses in patients having low immunity, or inflammatory and hyperstimulated immune responses, should be examined cautiously. It is possible that probiotic-mediated antagonism may deter some commensal bacteria or interrupt the signaling from indigenous microorganisms that are vital for health. Nevertheless, carefully designed, and broadly biologically safe, bioengineered probiotics can prevent health loss caused by drug-resistant infections (Figure 1).
restore feminine genitourinary health, and treat genetic metabolic diseases such as CD, ulcerative colitis, cardiovascular diseases, and urea cycle diseases. Some reports from initial clinical trials are encouraging. For instance, some recombinant products (e.g., LACTIN-V, https:// clinicaltrials.gov identifier NCT02766023; SYNB1020) (Table 1) have already been found to be safe and are ready for Phase II clinical studies in humans. Nevertheless, key challenges remain, primarily the selection of probiotic species, quality control, dose optimization, and lateral gene transfer from recombinant probiotics to other microbes. The acceptance of bioengineered probiotics as ‘drugs’ is possible provided they are tested by clinical studies for safety as per stringent guidelines of statutory bodies such as the US Food and Drug Administration (FDA), the European Food Safety Authority, or others. In conclusion, recombinant probiotics are potent futuristic alternative therapeutics to treat infectious and chronic diseases. We are optimistic about recombinant probiotics that are ready for testing in humans within competitive timeframes. As designer probiotics may restore health by delivering prophylactics with efficiency, ease, and site specificity, research should continue to explore human microbiota and develop potential strains as alternative therapeutics.
So far, most of the documented benefits of designer probiotics are from studies based on model animals. Whether designer probiotics could circumvent the side effects of antibiotic therapy should be a priority area of research. Developing probiotic formulations to prevent hospital- Acknowledgments acquired infections in critically ill trauma Financial assistance from SERB-DST (India) is patients would be a significant contribution acknowledged. of probiotic bioengineering to humans as 1 ICAR-Indian Veterinary Research Institute, Regional infection is the major cause of late deaths in Station, Palampur 176061, India 2 trauma patients. Healthy Aging Unit by Genomics & Biotechnology,
duly
Milano Medical (MMC), Milan, Italy
Recently, some biopharmaceutical and academic entrepreneurs have developed proprietary formulations: new classes of prophylactics containing live bioengineered probiotics and postbiotics to eradicate Clostridium difficile,
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*Correspondence: [email protected] (B. Singh). http://dx.doi.org/10.1016/j.tibtech.2017.04.001 References 1. Braat, H. (2006) A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn’s disease. Clin. Gastroenterol. Hepatol. 4, 754–759
2. Paton, A.W. (2012) Bioengineered microbes in disease therapy. Trends Mol. Med. 18, 417–425 3. Kumar, M. (2016) Bioengineered probiotics as a new hope for health and diseases: potential and prospects: an overview. Future Microbiol. 11, 585–600 4. Maxmen, A. (2017) Living therapeutics: scientists genetically modify bacteria to deliver drugs. Nat. Med. 23, 5–7 5. Mansour, N.M. and Abdelaziz, S.A. (2016) Oral immunization of mice with engineered Lactobacillus gasseri NM713 strain expressing Streptococcus pyogenes M6 antigen. Microbiol. Immunol. 60, 527–532 6. von Ossowski, I. et al. (2013) Using recombinant lactococci as an approach to dissect the immunomodulating capacity of surface piliation in probiotic Lactobacillus rhamnosus GG. PLoS One 8, e64416 7. Benbouziane, B. (2013) Development of a stress-inducible controlled expression (SICE) system in Lactococcus lactis for the production and delivery of therapeutic molecules at mucosal surfaces. J. Biotechnol. 168, 120–129 8. Ong, Z.Y. et al. (2014) Strategies employed in the design and optimization of synthetic antimicrobial peptide amphiphiles with enhanced therapeutic potentials. Adv. Drug Deliv. Rev. 78C, 28–45 9. Marcobal, A. (2016) Expression of human immunodeficiency virus type 1 neutralizing antibody fragments using human vaginal lactobacillus. AIDS Res. Hum. Retrovir. 32, 964–971 10. Lagenaur, L.A. (2011) Prevention of vaginal SHIV transmission in macaques by a live recombinant Lactobacillus. Mucosal. Immunol. 4, 648–657 11. Zhang, B. (2016) Recombinant Lactococcus lactis NZ9000 secretes a bioactive kisspeptin that inhibits proliferation and migration of human colon carcinoma HT-29 cells. Microb. Cell Fact. 15, 102 12. Carvalho, R.D. (2017) Secretion of biologically active pancreatitis-associated protein I (PAP) by genetically modified dairy Lactococcus lactis NZ9000 in the prevention of intestinal mucositis. Microb. Cell Fact. 16, 27 13. Danino, T. (2015) Programmable probiotics for detection of cancer in urine. Sci. Transl. Med. 7, 289ra84 14. Liu, K.F. (2016) Oral administration of Lactococcus lactisexpressing heat shock protein 65 and tandemly repeated IA2P2 prevents type 1 diabetes in NOD mice. Immunol. Lett. 174, 28–36 15. Chen, Z. (2014) Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity. J. Clin. Invest. 124, 3391–3406
Science & Society
Clusters in Industrial Biotechnology and Bioeconomy: The Roles of the Public Sector Jim Philp1,* and David E. Winickoff1 Government policies across the world seek to create clusters of
inexorable rise of antibiotic-resistant pathogens and the rather slow development of new antibiotics. Probiotics that deliver novel therapeutics efficiently and with site specificity are emerging living therapeutics that may transform existing paradigms of disease diagnosis and prevention.
Kluyveromyces lactis, and Pichia pastoris) are some of the prospective probiotics used for expressing heterologous genes encoding antimicrobial and anti-inflammatory biomolecules.
Designer Probiotics against Infectious Diseases As the efficacy of orally administered antigens is reduced during passage through the alimentary tract, probiotic-mediated drug delivery could be a promising strategy for administering multiple therapeutics (cytokines, antibody fragments, antigens, peptides, etc.) in situ at the site of infection, therefore circumventing the side effects associated with the systemic administration of drugs.
The normal microbiota are essential determinants of vital processes such as hematopoiesis, aging, immunity against infectious diseases, and behavior. Whereas the normal microbiota occa*Correspondence: [email protected] (.G. Papaioannou). sionally fails to protect the host against http://dx.doi.org/10.1016/j.tibtech.2017.04.011 pathogens, a perturbed gut microbiota is associated with inflammation, obesity, References 1. Karaberopoulos, D. et al. (2012) The theriac in antiquity. insulin resistance, diabetes, cardiovascu- For example, the vaccination of mice with Lancet 379, 1942–1943 lar diseases (CVDs), and neuropsychiatric recombinant Lactobacillus gasseri 2. Marx, V. (2015) Tissue engineering: organs from the lab. Nature 522, 373–377 disorders. NM713 expressing streptococcal M6 3. Aflalo, T. et al. (2015) Neurophysiology. Decoding motor protein (CRR6) protected them against imagery from the posterior parietal cortex of a tetraplegic The WHO has recently released a list of 12 streptococcus group A infections [5], human. Science 348, 906–910 4. Travis, J. (2015) Making the cut. Science 350, 1456–1457 antibiotic-resistant families of pathogenic and recombinant Lactococcus lactis 5. Brody, H. (2012) From an ethics of rationing to an ethics of bacteria, described as ‘priority patho- (LL-Thy 12) expressing human interleuwaste avoidance. N. Engl. J. Med. 366, 1949–1951 gens’, which pose a grave threat. Para- kin-10 (IL-10) provided relief from Crohn’s 6. Huxley, J. (1957) New Bottles for New Wine, Chatto & Windus doxically, the development of new disease (CD) [1]. Furthermore, L. lactis 7. Pepperell, R. (2005) Posthumans and extended experi- antibiotics has also slowed down, which strains that produce native (and pilinence. J. Evol. Technol. 14, 27–41 increases the demand for alternative ther- deleted) immunomodulatory surface pili8. Wehner, M.R. et al. (2014) Twitter: an opportunity for public health campaigns. Lancet 384, 131–132 apeutics. Expanding the efficacy of pro- ation appendages (SpaCBA) were found 9. Nabavi, S. et al. (2014) Engineering a memory with LTD biotics by introducing new genetic circuits to activate Toll-like receptor 2-dependent and LTP. Nature 511, 348–352 to deliver drug biomolecules is crucial. signaling in cell lines and to modulate 10. Wheeler, M.A. et al. (2016) Genetically targeted magnetic control of the nervous system. Nat. Neurosci. 19, 756–761 These recombinant probiotics, informally synthesis of anti-inflammatory cytokines 11. Min, B.K. and Muller, K.R. (2014) Electroencephalography/ called ‘designer probiotics’ or ‘probiotics (TNF-a, IL-6, IL-10, and IL-12) in human sonication-mediated human brain-brain interfacing tech2.0’, are poised to reduce the gap dendritic cells [6]. Another study reported nology. Trends Biotechnol. 32, 345–346 between the mounting antibiotic resis- recombinant L. lactis delivering therapeutance and the dearth of new antibiotics tic proteins at mucosal surfaces in murine [1–4]. models of human inflammatory bowel disScience & Society eases (IBDs) and human papillomavirus The science of probiotics, which was for- type 16 (HPV-16) [7]. merly confined to basic microbiology and food processing, has emerged in the In addition, epidemiological, experimenpostgenomic era of medicine and biology tal, and clinical evidence convincingly as a premier area of research towards shows that genitourinary microbiota domfunctional nutraceuticals, gastroenterol- inated by LAB protect the host against Birbal Singh,1,* Gorakh Mal,1 ogy, allergology, skin care, oncotherapy, bacterial vaginosis (BV) and sexually and Francesco Marotta2 psychoneuroendocrinology, and veteri- transmitted viral infections [9]. One connary applications. The lactic acid bacteria tributor to host protection is a group of Enhancing the functional reper- (LAB), bifidobacteria, Escherichia coli Nis- antimicrobial proteins including ribosotoire of probiotics is a promising sle 1917, and yeasts (Saccharomyces mally-synthesized bacteriocins, microapproach to cope with the cerevisiae, Saccharomyces boulardii, cins, peptides, and the type VI secretion 2
Biomedical Engineering Unit, First Department of Cardiology, Hippokration Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
Designer Probiotics: Paving the Way to Living Therapeutics
Trends in Biotechnology, August 2017, Vol. 35, No. 8
679
system (T6SS) of the gut and genitourinary and nasal tracts. These proteins are active at nanomolar levels and help protect against pathogens [8]. Langenaur and colleagues engineered vaginal-origin probiotic L. lactis and Lactobacillus jensenii to express HIV-inhibiting proteins for use as topical live microbiocides against HIV/AIDS and BV [9,10]. In addition to bacterial and viral infections, protozoan infections represent major health threats. The evidence that human gut microbiota elicit a protective immune response against Plasmodium spp. has led to the concept of evolving recombinant probiotics to cripple vector-borne diseases such as malaria, cholera, leishmaniasis, and trypanosomiasis. However, reports are currently scarce on recombinant probiotics against parasitic protozoa.
Anticancer Therapies Using Designer Probiotics The major challenge in oncology is a lack of availability of therapies against tumors with minimal toxicity to healthy tissues. Some approaches to treating cancer include chemotherapy, and prodrug delivery, targeted ultrasound, photodynamic therapy, biological therapy, and combinations of these strategies. Antitumor agents alone or in combina-
tion with radiotherapy or chemotherapy are toxic at therapeutic dosages. While reducing their dosage may lessen toxicity, their efficacy is also compromised. Certain bacteria, administered intravenously, colonize the hypoxic vicinities of tumors. In view of the ability of Bifidobacterium spp. and Clostridium spp. to preferentially proliferate in solid tumors, and currently aided by synthetic biology and genome engineering, there is renewed interest in using them as tumor-targeting vectors. Prodrug delivery methods, such as directed enzyme prodrug therapy (DEPT), use bacteria to express drug-converting enzymes (e.g., cytosine aminodehydrolase and nitroreductase). This approach may decrease the toxicity of drugs used in chemotherapy and eventually sensitize tumor cells to irradiation. Probiotics tailored to synthesize tumor-targeting peptides, induce cytotoxicity in tumor cells, or deliver chemotherapeutic drugs to remote tissues could be promising anticancer therapeutics.
colon carcinoma cells [11]. Pancreatitisassociated protein-I, expressed in a foodgrade recombinant L. lactis NZ9000, prevented experimentally induced intestinal mucositis in mouse models [12]. A proprietary oral rinse containing L. lactis, producing human trefoil factor 1 (hTFF1), ameliorated the severity of oral mucositis (OM) in a hamster model. The strategy is believed to be an effective tool for treating oral sores that develop following induction chemotherapy in patients suffering from head and esophageal cancer (Table 1). The programmable E. coli Nissle 1917, when administered orally, could detect hepatic metastasis signals in the urine of patients and is regarded as a live diagnostic probiotic [13].
Designer Probiotics and Metabolic Diseases
Endemic chronic diseases such as Alzheimer’s disease, autism, attention-deficit hyperactivity disorder, osteoarthritis, and stroke are increasing rapidly among human populations. Manipulating the gut ecosystem by recombinant probiotics expressing therapeutic biomolecules, fecal microbiota transplant, or phage Bioengineered L. lactis NZ9000-401- therapy might serve as alternative kiss1, producing KiSS1, a cancer-sup- approaches against lifestyle diseases. pressing peptide, is envisioned as a plausible anticancer probiotic by hampering When used as dietary supplements or migration and proliferation of human applied topically, designer probiotics
Table 1. Summary of Designer Probiotic and Postbiotic Formulations As Alternative Therapeutics Formulation (live component)
Manufacturer
Proposed applications
Actobiotics (L. lactis)
Intrexon, VA, USA
Targeted therapies against oral, gastrointestinal tract, metabolic, allergic, and autoimmune diseases AG013 attenuates OM and AG014 treats IBD through localized in situ delivery of anti-TNF-a Fab
AG013 (L. lactis secreting hTFF1)
ActoGenix N.V., Ghent, Belgium
Developing anti-TNF and bacterial formulation to prevent OM
CBM588 (Clostridium butyricum)
Osel Inc., CA, USA
Prevention of C. difficile infection and diarrheal disorders, restoring gut health after antibiotic therapy
LACTIN-V (Lactobacillus sp.)
Osel Inc., CA, USA
Restoring feminine genitourinary health by suppressing BV and viral infections
L. lactis
Oragenics, Tampa, FL, USA
Novel antibiotics, and proprietary probiotics for oral health of humans and pets
Multiple microbes
Seres Therapeutics, Cambridge, MA, USA
ActoBiotics drugs for treating diseases associated with dysbiosis
SYNB1020 (E. coli Nissle 1917)
Synlogic, Cambridge, MA, USA
Treating genetic metabolic diseases, IBDs including CD and ulcerative colitis, cardiovascular diseases, cancer, urea cycle disorders and hepatic encephalopathy (US Patent #9 487 464).
680
Trends in Biotechnology, August 2017, Vol. 35, No. 8
could support normal physiology and immunity to protect the host from infections, oxidative stress, inflammatory diseases, and autoimmune responses. For example, formulations that contain engineered probiotics producing human proinsulin and anti-inflammatory cytokines would provide a way to overcome the menace of diabetes. Orally administered L. lactis producing recombinant protein HSP65-6IA2P2 was found to prevent hyperglycemia, reduce insulitis, and increase glucose tolerance and regulatory
immune reactions in type 1 diabetes mellitus in nonobese diabetic mice [14]. Similarly, orally administered E. coli Nissle 1917 expressing N-acylphosphatidylethanolamines suppressed obesity in mice by regulating food intake. Because of similarities in eating behavior of mice and humans, the strategy might do well in humans [15].
Despite inconsistent and counterintuitive recognition for recombinant microbes and their metabolites, designer probiotics are envisaged as emerging living therapeutics for human and veterinary applicaConclusions and Perspectives Metabolic diseases and the surge of anti- tions. A combined therapy comprising an biotic resistance in pathogens are antimicrobial, anti-inflammatory, and
Ingeson/oral administraon
M Gut lumen
N
Designer probiocs
M D
Intravenous administraon (lyophilized bacteria)
N Urogenital healthcare:
Designer probiocs and peobioc matabolites
Restoring normal microflora, prevenon of allergy, an-HIV effects, protecon against BV/dysbiosis
Antumor acvies:
Ancancer effects:
Safe and selecve delivery of synthec genes to tumors; sensizing tumor cells to irradiaon
Producon of AMPs, CLAs, SCFAs, equol; removal of carcinogens; genotoxic matabolites
Anmicrobial effects:
DEPT/ CDEPT
Inhibion of angiogenesis, bacterial-mediated conversion of prodrugs to drugs in tumors
Food preservaon; inhibion of hospital-acquired infecons, IBD and parasic deseases, e.g., malaria
Disease diagnosis: Detecon of matastasis markers in body fluids and excreons
Tablets/ capsules
D
Gut epithelium Producon of proapoptoc or anproliferave factors (IL-10, TGF-β, SOD, catalase, SCFAs
serious public health concerns that require cost-effective inventive prescriptions. Microbes offer intriguing prospects for preventing illnesses that conventional medicines have failed to cure.
Managing obesity, depression, and cognive health
Sensing pathological/disease signals within gut; inhibiton of promitogenic and angiogenic acvies in cancer cells
Figure 1. A Diagrammatic Illustration of Health Benefits of Designer Probiotics (D) Instilled to Colonize the Mucosal Tissue. Synthetic biology and bioengineering are key research platforms to facilitate design and organization of the cell genome, create automated genetic circuits, and assemble large operons with integrated transcriptional and translational regulators. Designer probiotics are poised to benefit the host by eliminating pathogens, producing therapeutics (M) such as short chain fatty acids (SCFAs), antimicrobial peptides (AMPs), bacteriocins, enzyme inhibitors, cytokines, transforming growth factor-b (TGF-b), psychoactive compounds, conjugated linoleic acids (CLAs), and superoxide dismutase (SOD). Abbreviations: CDEPT, Clostridium directed enzyme prodrug therapy; DEPT, directed enzyme prodrug therapy; IBD, inflammatory bowel disease; N, normal microbiota.
Trends in Biotechnology, August 2017, Vol. 35, No. 8
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immunomodulatory repertoire of designer probiotics could combat infectious and metabolic diseases. Hence, probiotics that readily integrate with clinical prescriptions should be a prioritized area of research. Colonization of mucosal tissue could be a challenge for designer probiotics used as ‘drugs’, because of the intrinsic property of resident microbiota to resist instilled or invading microorganisms. Using humanorigin, commensal bacteria to deliver therapeutics could possibly resolve the problem. The safety of the end users and containment of exogenous genetic elements in recombinant probiotics are of concern. The likely probiotic-mediated rise of immune responses in patients having low immunity, or inflammatory and hyperstimulated immune responses, should be examined cautiously. It is possible that probiotic-mediated antagonism may deter some commensal bacteria or interrupt the signaling from indigenous microorganisms that are vital for health. Nevertheless, carefully designed, and broadly biologically safe, bioengineered probiotics can prevent health loss caused by drug-resistant infections (Figure 1).
restore feminine genitourinary health, and treat genetic metabolic diseases such as CD, ulcerative colitis, cardiovascular diseases, and urea cycle diseases. Some reports from initial clinical trials are encouraging. For instance, some recombinant products (e.g., LACTIN-V, https:// clinicaltrials.gov identifier NCT02766023; SYNB1020) (Table 1) have already been found to be safe and are ready for Phase II clinical studies in humans. Nevertheless, key challenges remain, primarily the selection of probiotic species, quality control, dose optimization, and lateral gene transfer from recombinant probiotics to other microbes. The acceptance of bioengineered probiotics as ‘drugs’ is possible provided they are tested by clinical studies for safety as per stringent guidelines of statutory bodies such as the US Food and Drug Administration (FDA), the European Food Safety Authority, or others. In conclusion, recombinant probiotics are potent futuristic alternative therapeutics to treat infectious and chronic diseases. We are optimistic about recombinant probiotics that are ready for testing in humans within competitive timeframes. As designer probiotics may restore health by delivering prophylactics with efficiency, ease, and site specificity, research should continue to explore human microbiota and develop potential strains as alternative therapeutics.
So far, most of the documented benefits of designer probiotics are from studies based on model animals. Whether designer probiotics could circumvent the side effects of antibiotic therapy should be a priority area of research. Developing probiotic formulations to prevent hospital- Acknowledgments acquired infections in critically ill trauma Financial assistance from SERB-DST (India) is patients would be a significant contribution acknowledged. of probiotic bioengineering to humans as 1 ICAR-Indian Veterinary Research Institute, Regional infection is the major cause of late deaths in Station, Palampur 176061, India 2 trauma patients. Healthy Aging Unit by Genomics & Biotechnology,
duly
Milano Medical (MMC), Milan, Italy
Recently, some biopharmaceutical and academic entrepreneurs have developed proprietary formulations: new classes of prophylactics containing live bioengineered probiotics and postbiotics to eradicate Clostridium difficile,
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Science & Society
Clusters in Industrial Biotechnology and Bioeconomy: The Roles of the Public Sector Jim Philp1,* and David E. Winickoff1 Government policies across the world seek to create clusters of