Team:IISER Bhopal/Poster


Poster: IISER Bhopal



THE BIG PIE: Beta Cells In Gut Produce Insulin using E.coli (Tackling Diabetes Without Injections!)
Presented by Team IISER_Bhopal iGEM Student

Team Member: Prerita Chawla, Rohan Dandavate, Shashaank G,
Harinarayan Kottala, Hitaishi Desai, Ruchir Gupta, Raibat Sarker, Sebin Abraham,
Priya Sharma, Kedar Bhosale, Archit Devarajan, Rishana Farin, Rita Abani.

Special Thanks to: Prajwal Bharadwaj, Soumyajit Chatterjee, Tanmay Bhore

iGEM Team Mentor: Anish Ganju

iGEM Team Primary PI: Dr Atul Kumar*
iGEM Team Secondary PI: Dr Chandan Sahi*
*Department of Biological Sciences, IISER Bhopal, Bhopal, Madhya Pradesh, India.

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Abstract
Over 85 million insulin-dependent diabetes patients who take insulin injections every single day. Our project THE BIG PIE aims to tackle diabetes without using insulin injections. We present an out-of-the-box approach to tackle diabetes from the root by creating beta islet-like cells within the body. Our therapy is based on the principle of transdifferentiation, makes use of three proteins that can be injected into gut cells, transforming them into beta islet-like cells. Our delivery vector, E. coli Nissle 1917, can be programmed to attach to crypt cells in the gut, followed by translocation of the three transcription factors - PDX1, MAFA & NGN3 - via the Type 3 Secretion System (a molecular syringe) converting our target cells into insulin-producing glucose-responsive beta-islet like cells. Finally, to prevent the release of our bacterial drug into the environment, we’ve also designed a kill switch.
We have designed iβeta as our capsule. Our bacterial drug will be enclosed inside the capsule and delivered to the target site. We have also modelled:
  • The entry of our capsule and its gut dynamics to ensure efficient delivery of our drug to the target niche.
  • The structure and function of our final proteins entering the target cells.
  • The functioning and optimisation of our kill switch.
Motivation
Over 460 million diabetics worldwide, of these more than 85 million patients are dependent on insulin injections for their survival. India is home to one in six diabetic patients across the globe, and 4 in 10 diabetics in India are dependent on insulin injections. However, Insulin injections are not easily accessible. They are inconvenient, painful and expensive, especially in the West. Regular usage of insulin injections can lead to lipohypertrophy, tissue damage and even infections.

While lack of insulin causes hyperglycemia and can lead to multiple organ failure, kidney damage, blindness and ultimately death. To tackle this dependency on injections, we’ve created THE BIG PIE - a therapy that can transform gut cells into insulin-producing, glucose-responsive beta-islet like cells that serve as a convenient and natural alternative to current therapies.
How is Diabetes Caused?
Diabetes mellitus is a chronic metabolic disorder caused due to insufficient insulin production in the pancreas or abnormal hormone regulation in the body. Insulin, a peptide hormone, produced in the beta islet cells inside the pancreas, is essential to regulate blood glucose levels in the body.
Type 1 diabetes patients have damaged or destroyed beta islet cells due to immune issues and certain Type 2 diabetes patients are insulin resistant, are usually dependent on injections for their insulin dosage.

Effector Construct
Construct with Polyprotein Sequence consisting of effectors - PDX1, MAFA and NGN3 and another sequence coding Tobacco Etch Virus(TEV) protease.

Studies have demonstrated that over-expression of PDX1, NEUROG3, and/or MAFA, can reprogram the fate of various non-β cell types into glucose-responsive insulin-producing cells.[1][2][8]

Polyprotein Sequence
  • MAP 20: Mitochondria associated protein is an export signal peptide sequence for T3SS, which helps guide the protein sequence through the T3SS pore.
  • PDX1: is a transcription factor required for early embryonic development of the pancreas and differentiation of endocrine cell types including β cells. Important for maintaining β-cell function.
  • NEUROG3: is a basic helix–loop–helix transcription factor family involved in the central nervous system and embryonic pancreas development.
  • MAFA: is a transcription factor that binds to the promoter region of the insulin gene and drives insulin expression. MAFA’s expression is important to regenerate functional β cells in regenerative approaches.
Interaction among PDX1, NEUROG3, and MAFA Transcription factors is essential. PDX1 regulates the expression of NEUROG3 and MAFA in the development and reprogramming processes.

The Protease Fusion Construct:
Expresses the TEV protease attached to a beta catenin degradation tag which ensures TEV protease is active only in the target niche and degraded in all other cells. The TEV protease cleaves the polyprotein at protease specific cleavage sites allowing effector proteins to function independently in the target niche.[3][4][13]
Attachment and Injection
We intend to use a novel delivery mechanism: Type III Secretion System (T3SS)8 -‘molecular syringe’ found on certain Gram-negative bacteria consisting of a basal body and filament. The T3SS will transport the linear polypeptide sequence consisting of effector proteins’ - PDX, MAFA and NGN3 into the crypt cells in the gut.
T3SS delivers Proteins in a two-step process:
  • The T3SS syringe inserts itself into the crypt cell membrane creating a pore.
  • With the help of a CesT chaperone, the protein sequence is unfolded and guided by MAP 20 the polyprotein consisting of PDX1, MAFA and NGN3 is translocated in linear form across the T3SS. On reaching the target cell’s cytoplasm, the protein sequence refolds into its native state. [6][7][9][14]

The sequence is then cleaved with the help of the TEV protease and transported into the nucleus where the 3 effector proteins PDX1, MAFA and NGN3 can exert their function and transform a gut cell into a beta-islet like a cell.[1][2] Both the protein unfolding and secretion are powered through a combination of ATPase activity and the proton motive force.
Delivery Vector
Our project iβeta is using an FDA approved probiotic bacteria E.coli Nissle 1917. E.coli Nissle is a nonpathogenic Gram-negative strain used in the therapies of many gastrointestinal disorders including diarrhoea, irritable bowel syndrome etc. and can reach the crypt cells in the duodenum of the small intestine.
T3SS our attachment system is only active in gram-negative bacteria. It was imperative to choose a system which is not only compatible with T3SS but also safe and compatible with our target niche inside the gut. Due to existing literature which supported the survival of E.coli Nissle in the small intestine, and the advantages of E.coli Nissle, we decided to choose it as our delivery vector.[11]

References
  1. Koblas, T., Leontovyc, I., Loukotova, S., Kosinova, L. & Saudek, F. Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors. Mol. Ther. Nucleic Acids 5, e320 (2016).
  2. Spit, M., Koo, B.-K. & Maurice, M. M. Tales from the crypt: intestinal niche signals in tissue renewal, plasticity and cancer. Open Biol. 8, (2018).
  3. Chen, X., Pham, E. & Truong, K. TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector. Protein Sci. 19, 2379–2388 (2010).
  4. Cesaratto, F., López-Requena, A., Burrone, O. R. & Petris, G. Engineered tobacco etch virus (TEV) protease active in the secretory pathway of mammalian cells. J. Biotechnol. 212, 159–166 (2015).
  5. New England Biolabs: Proteases.
  6. Wagner, S. et al. Bacterial type III secretion systems: a complex device for the delivery of bacterial effector proteins into eukaryotic host cells. FEMS Microbiol. Lett. 365, (2018).
  7. Walker, B. J., Stan, G.-B. V. & Polizzi, K. M. Intracellular delivery of biologic therapeutics by bacterial secretion systems. Expert Rev. Mol. Med. 19, e6 (2017).
  8. Jin, Y. et al. Enhanced differentiation of human pluripotent stem cells into cardiomyocytes by bacteria-mediated transcription factors delivery. PLoS One 13, e0194895 (2018).
  9. Preston, G. M. Metropolitan microbes: type III secretion in multihost symbionts. Cell Host Microbe 2, 291–294 (2007).
  10. Salema, V. et al. Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies. PLoS One 8, (2013).
  11. Costello, C. M. et al. 3-D intestinal scaffolds for evaluating the therapeutic potential of probiotics. Mol. Pharm. 11, 2030–2039 (2014).
  12. Piñero-Lambea, C. et al. Programming Controlled Adhesion of E. coli to Target Surfaces, Cells, and Tumors with Synthetic Adhesins. ACS Synthetic Biology 4, 463-473 (2014).
  13. Spit, M., Koo, B. & Maurice, M. Tales from the crypt: intestinal niche signals in tissue renewal, plasticity and cancer. Open Biology 8, 180120 (2018).
  14. Munera, D., Crepin, V., Marches, O. & Frankel, G. N-Terminal Type III Secretion Signal of Enteropathogenic Escherichia coli Translocator Proteins. Journal of Bacteriology 192, 3534-3539 (2010).
Kill Switch
We propose a single gate phosphate regulated kill switch that is active at low phosphate concentrations and kills the modified bacteria post egestion and entry into the environment (has low phosphate conditions).



Expression
Toxin - Colicin E2 DNase domain "miniColicin" (BBa_K1976048), is expression under constitutive promoter Pbad. This toxin can degrade host DNA whilst posing little threat to the gut microbiota. Anti-toxin - immunity protein IM2 (BBa_K1976027). At high phosphate concentration, IM2 binds to miniColicin’s DNAse domain and prevents it from functioning. IM2 is expressed under cI lam promoter (BBa_R0051).

Killing Mechanism
High phosphate Concentration (small intestine): Phosphate regulated PphoA promoter is inactive, allowing the bacteria to function.
Low phosphate concentrations (in the environment): PphoA promoter is activated and transcribes the repressor protein - cI repressor from phages. This repressor binds to the cI lam promoter and renders it inactive, thus stopping the translation of anti-toxin inside the cell. The constitutively expressed toxin increases in concentration over time. It kills the bacteria which dies in the low phosphate environment by the time it is egested and released into the environment.
Antibody - mediated Adhesion System

We intend to display a Single domain Antibody (SdaB) with a β intimin base attached to E.coli Nissle surface.[10] The Sdab antibody corresponds to Lgr5 antigen present on the surface of the target cells - crypt cells in the gut.[12][13] The antibodies ensure that the delivery vector binds to the right type of cells. The single-domain antibodies from camelids bind to β intimin a surface protein derived from EHEC. The display of the antibody on beta intimin guarantees a stronger affinity for the target cells.
THE BIGPIE
Our project is a proof of concept that we intend to complete in the lab with SIEC-eLEE5 (E.coli K-12 containing T3SS) as our chassis. We will clone all proteins and constructs into the E.coli K-12 further test it with a suitable small intestinal cell line and ascertain the functioning of our beta islet like cells and their insulin production.

Future Wet Lab
  • The experiments followed with SIEEC-eLEE5 will next be tested with E.coli Nissle, an FDA approved, Gram-negative probiotic bacteria that can survive in the small intestine.
  • The next step would be to test the transformation of the gut cells into beta islet-like cells using modified E.coli Nissle with human organoids.
Delivery Capsule iβeta

THE BIG PIE bacteria are then enclosed in a delivery capsule called iβeta. iβeta has an HPMC enteric capsule dissolves at a pH of 5 while the duodenum has a pH of 5.5 in the fasting state. The capsule can be ingested before a meal and is ejected from the stomach.
iβeta begins dissolving in the duodenum releasing the PLGA microspheres that are coated with PEG. The PEG microspheres penetrate the mucous membrane of the target niche. Thus releasing the bacterial drug near the target site, which is the epithelial layer below the mucus that contains the crypt cells.

CAT model for Drug Delivery (Mathematical Modelling)
Objective: To get an approximate idea about the absorption constant required for our drug to reach the mucus layer. This constant can be related to a parameter that can be determined experimentally (Peff).
Assumption:
  1. Small intestine is divided into seven compartments with equal residence time.
  2. Maximum absorption occurs in the first 2-3 compartments (duodenum and early jejunum).


Results: Linear transfer kinetics shows the absorption constant must be minimum 3 hour-1 for efficient drug release.
Clinical Trials
To test the efficiency, safety and efficacy of our drug. We’ve developed a series of fool proof clinical trials.

Rodent Trials: 2 sets of identical rodents will be split into the Experimental group and Control group.
Type 1 Diabetes: The trial will be performed on streptozotocin mice and Non -obese Diabetic (NOD) mice.
Type 2 Diabetes: Zucker Diabetic Fatty Rats.



Human Trials
Entrepreneurship
iβeta is an affordable bio-therapeutic in capsule form that does not require special manufacturing facilities. iβeta can facilitate natural insulin production in-vivo, in a glucose-responsive manner, overcoming hypoglycemia associated with conventional therapies. It can help diabetic patients lead a normal life & is not likely to have adverse effects on long-term usage as seen in the current therapy (e.g. lipohypertrophy associated with insulin injections).
iβeta is user-friendly and a low-frequency drug, it needs to be administered a maximum of twice a week.

The current Indian diabetes market is valued at INR 145 billion and is growing at a CAGR of 16.7%. iβeta is expected to enter the market in ~12 years when the estimated market size for diabetes is INR 925 billion.


PS: For our first step towards taking iβeta to the people. We have participated in CurveBall-2020, an Institute level entrepreneurial competition and have been offered a space in our in-house incubation cell for a period of one year.
Modelling Results
Structural Modelling
Objective: to understand and verify that the function of the 3 TFs is not lost/altered during this process. In other words, we need to make sure that the active domains of our TFs are unaffected by the polyprotein formation and cleaving process.

Alignment of PDX1 (with residues (magenta) and without residues (grey))
RMSD: 0.879 (for active domains only)
Alignment of MafA (without residues (firebrick) and with residues (yellow))
RMSD: 1.501 (for active domains only)
Alignment of Ngn3 (without residues (red) and with residues (cyan))
RMSD: 0.774 (for active domains only)

Result: Alignment of the active domains predict that the additional residues don’t affect the functionality of our effectors.

Kill Switch
Initial design: Consisted of antisense RNA complementary to antitoxin expressed downstream of the PphoA promoter. The toxin Colicin E2 and antitoxin IM2 are expressed constitutively. In low phosphate concentration, the PphoA promoter is active, the antitoxin expression is suppressed by antisense RNA, increasing the concentration of free toxin.
After modelling, we realized that the kill switch is not functional at low phosphate concentration.


Final Design: Cl repressor is expressed downstream of the PphoA promoter. IM2 antitoxin is expressed downstream of the cl lam promoter. At low phosphate concentration, cl repressor is translated and represses cl lam promoter, so IM2 concentration is suppressed, and free toxin concentration increases. Prediction by mathematical modelling showed us a useful kill switch.

Optimisation: Toxin Colicin E2 replaced by Colicin E2 DNase domain "miniColicin".

References
  1. Lengyel, M.; Kállai-Szabó, N.; Antal, V.; Laki, A.J.; Antal, I. Microparticles, Microspheres, and Microcapsules for Advanced Drug Delivery. Sci. Pharm. 2019, 87, 20. http://dx.doi.org/10.3390/scipharm87030020
  2. Min Liu, Jian Zhang, Wei Shan, Yuan Huang (2015) Developments of mucus penetrating nanoparticles. Asian Journal of Pharmaceutical Sciences. https://doi.org/10.1016/j.ajps.2014.12.007
  3. Y.Y. Wang, S.K. Lai, J.S. Suk, et al. Addressing the PEG mucoadhesivity paradox to engineer nanoparticles that “slip” through the human mucus barrier Angew Chem Int Ed Engl, 47 (2008), pp. 9726-9729. https://doi.org/10.1002/anie.200803526
  4. Evonik Industries.
  5. 5 Different Types of Capsules - Pill Capsules Shell Guide
  6. Capsule Size Chart
  7. Encapsulation technology to protect probiotic bacteria
Integrated Human Practices

Dr Ishaan Gupta
Changes in effector protein construct. Initially the proteins were being translated and translocated into the target cells separately. Dr Gupta pointed out the inconsistency in the translation rates of the proteins and hence we decided to translate them 3 proteins as a single polyprotein sequence and translocate them together to maintain a 1:1:1 stoichiometric ratio.


Dr. Varun Chaudhary
Advised us to fuse our protease with beta catenin as a our degradation tag to maintain specificity in our target niche.


Dr Padma Devarajan
Guided us on creating a suitable capsule for efficient delivery of the bacteria drug to the target niche.


Dr Ravindra Shukla
Guided us on creating useful clinical trials that would be relevant to our project implementation.


Dr Cansu Uluseker
Provided us with relevant data about the parameter values required to model the function of the pPhoA promoter that we are using in our kill switch.
Contributions
TEV protease (BBa_K1639008) can be delivered with the genes of interest as a fusion protein to construct multi-gene circuits with a single vector.[1] TEV protease can be used to cleave fusion proteins and to couple the translational rates of different factors to each other, enabling a relatively stricter stoichiometric control over existing systems.

Exendin-4 (BBa_K3096030) can suppress the PI3K/Akt/mTOR pathway and inhibit the enzalutamide induced invasion and migration of prostate cancer cells.[2] Exendin-4 and enzalutamide treatment might be beneficial for patients with advanced prostate cancer.[3]

CesT (BBa_K2871004) can sense the activation of the Type III Secretion System via the release and translocation of Tir, one of its substrates.[4] [5] It can then transduce this information to the trans-acting post-transcriptional regulatory factor CsrA through sequestration, hindering CsrA’s ability to act on and silence its target mRNA.[5]

Additionally, we have codon-optimized the existing sequences of these parts for expression in Escherichia coli.

We have also created a Patent guide accessible on our to help all Indian iGEM teams who are interested in patenting and scaling up their project beyond iGEM.


References
  1. Chen X, Pham E, Truong K. TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector. Protein Sci. 2010;19(12):2379-2388. doi:10.1002/pro.518
  2. He W, Li J. Exendin-4 enhances radiation response of prostate cancer. The Prostate. 2018;1–9. https://doi.org/10.1002/pros.23687
  3. He W, Shao Y, Yu Y, Huang W, Feng G, Li J. Exendin‐4 enhances the sensitivity of prostate cancer to enzalutamide by targeting Akt activation. The Prostate. 2020;1–9. https://doi.org/10.1002/pros.23951
  4. Ye, F., Yang, F., Yu, R. et al. Molecular basis of binding between the global post-transcriptional regulator CsrA and the T3SS chaperone CesT. Nat Commun 9, 1196 (2018).
  5. Elbaz N, Socol Y, Katsowich N, Rosenshine I. 2019. Control of type III secretion system effector/chaperone ratio fosters pathogen adaptation to host-adherent lifestyle. mBio 10:e02074-19
Sponsors
Acknowledgements
  • SOUMYAJIT CHATTERJEE
  • TANMAY BHORE
  • SUKRIT CHAWLA
  • SAKSHAM JAIN
  • ANURAG YADAV
  • ANURAG CHITTAWAR
  • ISHITA GUPTA
  • ASHWIN ANANTHANARAYANAN
  • MANAS M. JOSHI
  • ADITI CHAUDHARI
  • AMEY DANOLE
  • SONAM KULKARNI
  • S. GANGOTHRI
  • PRAJWAL BHARADWAJ
  • YATHARTH PAL
  • KANISHK HARDE
  • SHUBHAM GAVHANE
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  18. Team:IISER-Pune-India/Model/regulatory
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