Team:IISER Bhopal/Design
Design
The BIG PIE is a biological delivery system created to allow insulin production in-vivo within the body. The objective of BIG PIE is to compensate for the decreased/defective β-islet cells in the pancreas of diabetes patients by reprogramming the gut cells into β-islet cells and eliminating the need for external sources of insulin. We intend to package our modified bacteria as a capsule which creates our final product iβeta.
The design of this project has been refined constantly to create a delivery system which is effective, reliable and most importantly safe by design.
Click on the individual parts of this Bacteria to know more!
Representative Bacteria
Representative bacteria shows all constructs we intend to clone into our bacteria delivery system.
- Plasmid1(left) : Polyprotein sequence with 3 effector proteins and protease fusion sequence have been cloned for reprogramming into β cells.
- Plasmid2(right): Kill switch construct needed to kill bacteria outside the body.
- Needle Like T3SS injection for penetrating and attaching to gut cell membrane.
- VHH antibodies specific to the target cell.
Project Principle
Our project is based on the principle of “transdifferentiation”. We intend to reprogram a set of the newly differentiated intestinal crypt cells in the duodenum (gut) to β-islet-like cells, capable of glucose-responsive insulin secretion.
Previously transdifferentiation has been performed in human organoids and mice models with the aid of adenovirus vectors: a viral delivery system. Due to the pathogenic nature of viruses and the risks associated with them (mutagenesis, oncogenesis and immunogenicity), we decided to replace the viral vector with a probiotic bacterial vector that is safely accepted into the host.
Keeping in mind these aspects of our project, our design stands on 4 pillars which form the backbone of our work.
Our project iBETA 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.
E.coli Nissle is classified as a probiotic due to its lack of defined virulence factors along with the expression of fitness factors such as microcins, and iron uptake systems, and proteases which may support its colonization of the human gut. EcN exhibits a semi-rough lipopolysaccharide (LPS) phenotype and serum sensitivity and does not produce known toxins. EcN colonizes the intestine within a few days and it remains as colonic flora for months after administration.
Our literature reviews showed us that several cell types in the stomach, gut, liver could be transformed into beta islet-like cells capable of producing insulin. But we chose the gut because it is easily accessible. Our bacterial drug can reach the gut by oral consumption via capsules. The well-characterised environment of the intestine can easily host our bacteria and will show a long term result to our treatment. The crypt cells in the gut were part of the epithelium which made it relatively easy to access, most importantly they are young and developmentally flexible stem cells, that can be easily reprogrammed into beta islet-like cells by our bacterial delivery system using the principles of transdifferentiation.
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 cloned in E.coli Nissle will transport the linear polypeptide sequence consisting of effector proteins’ - PDX, MAFA and NGN3 into the crypt cells in the gut.
Studies have demonstrated that overexpression PDX1, NEUROG3, and/or MAFA, can reprogram the fate of various non-β cell types into glucose-responsive insulin-producing cells. Here’s an explanation of every part of our plasmid construct.
MAP 20
Mitochondria associated protein is a 20 amino acid long export signal peptide sequence for T3SS, which helps guide the protein sequence through the T3SS pore.
TMV Protease Substrate
Protease cleavage sites are required to separate and activate the transcription factors in the cytosol of the effector cells.
PDX1
is a homeodomain transcription factor and is required for early embryonic development of the pancreas and differentiation of endocrine cell types including β cells. It plays a critical role in maintaining β-cell function.
NEUROG3
is a member of the basic helix–loop–helix transcription factor family involved in the central nervous system and embryonic pancreas development.
MAFA
also known as RIPE3b1, is a member of the MAF family of basic leucine zippers. It is a transcription factor that binds to the enhancer/promoter region of the insulin gene and drives insulin expression in response to glucose. For regenerative approaches, MAFA’s expression is also important to regenerate functional and mature β cells from pluripotent stem cells.
Interaction among PDX1, NEUROG3, and MAFA Transcription factors PDX1, NEUROG3, and MAFA are also mutually interacted/regulated during the pancreatic developmental process. PDX1 regulates the expression of NEUROG3 and MAFA in the development and reprogramming processes.
As per literature the proteins PDX, MAFA and NGN3 have been able to survive in human organoids and gut cells in mice. There is no protease action on these proteins as they are pre-existent in the gut, in lower concentrations. The presence of these transcription factors at high concentrations will influence the regulatory pathways to produce insulin in the reprogrammed gut cells while ensuring survival of the transcription factors as they are not considered foreign in the gut cells.
There were a number of strategies thought of for the targeting system. But we finally decided on a three-step targeting system. To ensure absolute accuracy and specificity in the attachment of our delivery vector to the target cell. It was imperative that transdifferentiation occurs specifically and only in the undifferentiated crypt cells of the duodenum.
While drug delivery design is not directly connected to the project design. It is essential to our targeting system prior to cell binding. Our therapeutic is enclosed in a capsule that forms an essential part of our targeting system to ensure the efficient and safe delivery of the bacteria to our target cells. Our bacteria are enclosed in an HPMC capsule which is pH sensitive and prevents the release of the bacteria in the stomach while our microspheres allow targetted entry into the crypt regions present below the epithelial mucous. To find out more about our capsule targeting system visit the implementation page.
Antibody specific to Crypt Cells We intend to display a Single domain Antibody (SdaB) with a β intimin base attached to E.coli Nissle surface. The SdaB corresponds to the Lgr5 antigen exposed on the crypt cells(CBCs) in the gut. The antibodies ensure that the delivery vector binds to the right type of cells. The single-domain antibodies from camelids (nanobodies or VHH) bind to β intimin a surface protein derived from EHEC. The display of the VHH on intimin guarantees a stronger affinity for the cell-specific antigens. The sdABs also survived better on the E.coli surface with very little proteolysis or cell toxicity. Thus this antibody display on E.coli surface specific to antigens on crypt cells is the first step that ensures accurate binding of the delivery vector to the crypt cells.
The strategies we are currently exploring involve displaying antibodies against cell-type-specific markers on E. coli’s surface10, displaying a cell-specific protease-sensitive T3SS inhibitor on E. coli’s surface, and fusing our effectors to C-terminal cytoplasmic retention signals downstream of the cleavage site of a cell-specific protease.
Our final regulation step takes advantage of a pre-existing Wnt signalling pathway present mostly in the crypt base cells which is our target cell. We intend to clone a protein protected by the Wnt signalling pathway, β-catenin along with the protease TEV Protease (S219N) can cleave our polyprotein sequence and seperate the Transcription factors PDX1, MAfa and NGN3 in the cell cytosol.
In most cells β catenin(and attached protease) is degraded by the environment and hence the polyprotein sequence will not be active. However, in the crypt cells, the Wnt signalling pathway protects the β catenin, and the tagged protease can survive.
Since β catenin is a transcription factor usually found in the nucleus. We are also placing an NES(Nuclear Export Signal) at its C-terminus to retain it in the cytosol of the effector cell. This allows the cleavage of the polyprotein sequence in the cytosol, transport of the transcription factors to the nucleus and activation of downstream signalling required for cellular reprogramming.
Safety has been a primary concern while creating Genetically modified organisms (GMOs). GMO usage has led to several claims and arguments about its unwarranted impacts on both the consumer and the surroundings. Keeping these safety aspects in mind, we have made some design considerations in our project which will ensure the safety of the gut microbiome, the host and the environment in which the pill contents will be released.
To avoid GMO release into the environment we propose a single gate phosphate regulated kill switch that is active at low phosphate concentrations and kills our probiotic bacteria by the time it is egested and enters the environment (which has low phosphate conditions).
Expression
To kill the engineered probiotic, the Toxin - Colicin E2 DNase domain "miniColicin" (BBa_K1976048), is expressed under a constitutive promoter Pbad. This toxin can degrade host DNA whilst posing little threat to the gut microbiota.
In the bacteria’s functional state, the toxin activity is inhibited by expressing the anti-toxin - immunity protein IM2 (BBa_K1976027) -which has a high affinity for miniColicin’s DNAse domain and prevents the toxin from functioning. The anti-toxin is expressed under promoter cI lam (BBa_R0051).
Killing Mechanism
The kill switch is activated at low phosphate conditions. At high phosphate concentrations in the small intestine, the PphoA promoter is inactive, allowing the bacteria to function. At low phosphate concentrations post egestion of the bacteria and removal from the body, PphoA promoter is activated and transcribes a repressor protein called cI repressor derived from E.coli phage lambda. 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.
The human gut is colonised by millions of bacteria. A major concern with our proposed probiotic THE BIG PIE, is its release into the small intestine, which could cause in horizontal gene transfer, resulting in lab made plasmids entering the other natural bacteria present in the gut microbiome.Keeping this in mind, we propose to use chromosomal integration of our parts in the final probiotic bacteria that we will introduce into the pills. The integration of our genetic circuits and cassettes into the chromosome will remove any possibility of horizontal gene transfer.