For human practice, we gathered basic information with a public survey on diabetes and a public survey on diet. Our survey sample was 70 high school students in South Korea. In the public survey on diabetes, our team could find out students generally merely had overall concepts of diabetes. Students’ view toward diabetes patients were negative (lack confidence, have an idle habit, lack self-control) to some degree. In addition, they also had an image that diabetes is caused by overeating and lack of exercise even though some diabetes partially occurs genetically. Moreover, students believed they had low possibilities to get diabetes; in fact, teenagers’ incidence of diabetes is currently increasing.
As non-specialist high school students, we thought it was important to thoroughly learn about subjects relevant to our project. Hence, we constantly reached out and exchanged opinions about with experts from suchfields. In fact, our discussion was not limited to the iGEM project--we often engaged in fervent dialogue about general biology and pharmaceuticals, that we often, unintentionally, took hold of the experts for too long. Here, we want document more about these general discussions (Discussions regarding our iGEM project can be found in the integrated Human Practices section in detail). Other than our project, we delve into two topics in particular: GMO in biomedical industry and insulin therapy for Diabetes.
GMO in biomedicine
On July 13th, we visited Ildong Pharmaceuticals to meet four researchers, including Tae Yoon Kim. First, we presented Before going to the interview, we sent five questions relevant to our project, mainly about the use of probiotics bacteria, and Mr. Kim gave a presentation to answer our questions.
Q1. What is the key technology in development of probiotics drug? Can you introduce some of your product?
The most important process is to screen appropriate bacteria and to test the productivity and efficiency of incubation. Furthermore, it is extremely hard to keep the bacteria alive when administered in human body. In fact, one of our probiotics drug uses dead cells. Inside microbiome, harmful bacteria can both weaken the immune response and cause too much immune response--inflammation. This product causes negative signals to cancel out this effects.
Q2. We have recently heard that microbiome based drug is in development. Which drugs are being developed? What are some problem in regulations?
The microbiome based medications causes change in the gut microbiome. They normalise the the environment inside the gut, and has the potential to cure or prevent diseases such as cancer and diabetes. However, in South Korea, there is no valid regulation about microbiome medication; it is virtually impossible to develop such medication. Meanwhile, though not official yet, guidelines are coming out in US.
Q3. Is there any medication that uses gene technology? Can you introduce some of your products?
Unfortunately, we don't have any gene tech-based drugs. The effectiveness of GMO drugs isn't as good enough to forgo the potential risks. Meanwhile, we don't really call protein based drugs produces by GMO as GMO drugs. We use the protein only because of the problems related to antibiotics resistance.
Q4. Which capsule do you use to deliver probiotics/ drug to intestine?
We use enteric coated capsules, which prevent the drug from melting in stomach. Lactoby enteric coated capsule, a commonly used capsule, is made up of plastic, fiber, long chain fatty acid, and glycerol. Another option is enteric coated tab, which is in a form of powder. Tab is made by drying and compressing the mixture of the bacteria, lactose and water. This process, however, involves high temperature heat which can kill the bacteria. Since it is important to keep the probiotics alive, it is more common to use enteric coated capsule.
Q5. Can you explain the process of getting approval for probiotics based drugs?
Probiotics medicines aren't easily approved. It's only in Japan where probiotics is regarded as a medicine. In most of the countries, probiotics is regarded as food. The process requires four clinical trials.
Furthermore, Seunghee Jeong, a researcher from Korean Yogurt Central Research Instituiton offered important advises about using probiotic bacteria as a potential drug delivery system as well as regulations around GMO in general through e-mail. We translated her advice into English, so that other iGEM teams could learn about the processes necessary to industrialise GMO.
In order to use gut bacteria as a host to produce various materials such as enzyme and protein, it is necessary to understand the regulation system around the safe usage of probiotics. For example, though Lacto bacillus is a food grade bacteria, using the strain for food as GMO is extremely limited.
The regulation around GMO for food differs greatly by country. While the restrictions are relatively lax in America, Europe and Oceanis abide by a much stricter regulations. But generally speaking, in Food grade expression system, the chassis used for engineering purpose must be safe and stable. Furthermore, it should use selectable marker that is compatible with food, which excludes antibiotic resistance marker. Furthermore, both the chassis and the expression system should not produce toxin or allergen, and the genetic modification should be done by self replication. Most importantly, the food grade modified gut bacteria should be stable in Human microbiome.
The definition of GMO differs by country as well. In USA, "genetic modification" is the act of modifying DNA sequence using certain technique and the approval is done by the characteristic of the chassis. In fact, foods produced by bacteria engineered by synthetic DNA technology is called "bioengineered food," not GMO. According to American principles, the genetically modified gut bacteria is classified as "GRAS," and is widely commercialised. On the other hand, in Europe, all bacteria modified in vitro, which didn't arise naturally, are regarded as GMO. In Oceanis, only the bacteria created by certain bio technologies are regarded as GMO.
In South Korea, all bacteria modified in vitro are regarded as GMO. KFDA is in charge of the regulations. Hence, your project seems to be inappropriate as a food, but is eligible as biomedicine.
Insulin therapy for Diabetes
On June 9th, we visited Hanlim Univ. Gangnam Seongshim Hospital and met Dr. Choi. During the interview sessions, we discussed about the methods and medications of treating Diabetes. He even showed use the insulin pen that patients use to inject insulin analogue subcutaneously. The following summarises the highlights of our dialogue.
Insulin therapy uses three types of insulin analogues: Short acting bolus insulin (administered at each meal), Long acting basal insulin (Once a day, functions for more than 24 hours), and pre-mixed insulin. Injections take place either four times or twice a day. An alternative for manual insulin injection with needles is insulin pump, which is very expensive. For Type I Diabetic patients, insulin therapy is necessary.
Side effects of insulin therapy include increase in weight and fat. Also, because of insulin resistance the dose of insulin should be increased every once a while.
Common oral medications for Diabetes are SU drug, Metformin, Glp-1 agonist, DPP4 inhibitor, TZD, and alpha glucosidase inhibitor.
On June 17th, KDA (Korean Diabetes Association) Workshop to listen to lectures about the treatment of Diabetes from top professors. Our of six lectures, we gained most relevant knowledges from two particular lectures: "Effective us of Anti-diabetic oral medications in combination therapy" and "Insulin therapy."
Effective us of Anti-diabetic oral medications in combination therapy:
70% combination therapy increases efficacy for a few reasons. First of all, it decreases the side effects because the doses for each drug is much lower. Secondly, the use of pill and capsule increases the adherence to the therapy as compared to invasive insulin injections.
Disadvantage of insulin therapy include risk for hypoglycemia, cardiovascular risks, cost, and inconvenience.
The most recommended combination is Metformin, SU] (Sulfunylurea), and DPP4 inhibitor.
Insulin therapy:
Insulin therapy that involves basal insulin and parandial (bolus) insulin is the most standard therapy because it allows for flexible injection schedule.
Pre-mixed insulin is made up of 50% insulin and 50% parandial insulin. Pre-mixed insulin has limited flexibility.
The use of GLP-1 agonist along with basal insulin is highly recommended. Glp-1 can lead to glucose dependent parandial insulin secretion. In the recent LEADER study, the use of glp-1 decreased all cause death significantly. Basal/glp-1 agonist combination doesn't cause much increase in body weight and hypoglycemia.
The side effects of glp-1 include pancreatitis, thyroid cancer, and adverse G1 events, or Nausea.
During Q&A session, we asked about the reason why insulin can not be administered orally and for potential substitution for subcutaneous injections:
Insulin is easily degraded by gastric acid, and cannot go through the intestinal mucous.
Some alternative methods are spray type medication, insulin patch, inhaler (Afreza), and Insulin pump. However, Insulin pump is not a complete close loop and can be more adverse to patients who are not good at taking care of the schedule. Some automatic, smart insulin pump also exist...but they are very expensive
On September 11th, we visited Eli Lilly & Company, a leading pharmaceutical company that produces insulin analogues. We were given a presentation about the history of insulin production, which started from pig and ultimately ended in genetically modified organisms, and Lilly's involvement. Her presentation spanned to the current developments of insulin analogues, about the structure and function of different insulin analogues that work for differing amount of time and for diverse purposes.
Other
Lastly, we also visited ISIS patent office to meet Hyung Sang Kim, a patent attorney. We explained to him our plasmid design and discussed about the possibility of patenting the minicell-based delivery system that we have devised. He told us that we should apply the patent for the plasmid itself, and that we need evidence to prove that our design includes unique considerations and is the most optimal design for the mooted purpose. We decided to go through the patent process after the iGEM competition, so that we have enough data to prove the practicality of our method.
Our project, under the aim of developing oral delivery methods of insulin, has gone through significant evolution as we met advisors and stakeholders. Every in-depth conversion with these key individuals allowed us to find out potential hazards or sloppy holes of our design. At the end of the day, we always thought of a way to integrate each of these lessons into our project design. Below, you can find out our original project design, our final project design as well as the advices that we received along the evolution.
Our Original Project Design
Originally, our project was named the ECOCO system which involved two method of oral delivery of insulin. The first half involved the E.coli Coworkers, which is a solution that involves a gut bacteria that secretes basal level of insulin after colonizing human gut. The other half involved the E.coli Coriers, which are minicells that deliver bolus insulin after each meal--the part that survived from our initial design. You can learn about the details of original plasmid construct and design from the presentation files (see above) that we used to introduced our project during first few interviews.
YS NaFamily of a Type 1 Diabetes Patient... and Kyuhee Jo's close friend
Minkyu ChoiA family doctor at Hanlim Gangnam Univ. Hospital
KDA WorkshopDiabetes workshop held by Korea Diabetics Association
Taeyoon Kima senior researcher from Ildong pharmaceutical company
Hyungsang KimA patent attorney at ISIS inc.
LillyPharmaceutical company that produces Insulin analog
To: The Evolution of Our Project Design
YS Na
I have a brother who is afflicted with Type I Diabetes. He has to inject insulin every single day for his entire life. It seems to hurt really much whenever he poke his own belly with a needle. My mom had to force him when he was younger. Even now, he often procrastinate or forget insulin injections. He's going to a boarding school next year and I am worried whether he would be able to take care of the injections himself.
Kyuhee Jo
Gosh, I didn't know! That must be so exhausting. I feel so sorry, I hope I could help your brother one day.
2 years later, Kyuhee Jo became senior at highschool. She assembled an iGEM Team from her school HAFS, and the team started to brainstorm about various project ideas. Diabetes was one of the various ideas that came out as a potential subject of dissertation. In order to delve more deep into the subject, iGEM HAFS received advice from medical practitioners. Among them was Dr. Mikyu Choi, who provided essential information that led to the formation of the project idea.
Minkyu Choi
In fact, this is not a problem exclusive to her brother. All type 1 Diabetes patients and a number of type 2 Diabetes patients get Insulin therapy as a necessary part of their efforts to overcome diabetes. However, since insulin is a protein readily degradable along the human digestive tract and is a very heavy molecule that does not penetrate epithelial cells well, it is basically impossible to deliver insulin orally. That's why patients have press the needles deeply into the fat layer under the skin and inject the insulin analog in painful way--not even once, but four times a day.
Minkyu Choi
Minkyu Choi
Normal people conserve certain level of basal insulin during the day, which is interrupted by spikes of insulin secretion after each meal. During insulin therapy, the insulin analogs function to mimic this pattern of plasma insulin level. Long acting insulin analog is injected once a day to increase basal level of insulin. Bolus doses of short acting insulin analog is injected before each meal to control glucose spikes after eating food. This combined...is four times a day.
Minkyu Choi
But this natural aversion towards invasive therapy is more serious of a problem than you may initially assume. Along the course of Type 2 diabetes, there comes a time in which Insulin Therapy is necessary to take control of the disease. However, about 70~80% of Type 2 diabetic patient refuses Insulin therapy due to their fear towards injections. This can rapidly worsen the disease. Furthermore, since insulin injection is very inconvenient and takes much time, patients have hard time regularly injecting the insulin analogs at right time. When they do forget, or forgo the right time, the result is often hyperglycemia--increased blood glucose level, which can lead to serious complications.
Team HAFS
Thankyou for providing important insights about insulin injections! It sound like a very serious problem, even more than what we previously thought of. Maybe we should seriously consider about developing oral delivery method of insulin using synthetic biology techniques through iGEM.
Once the subject was set, team HAFS started to design a synthetic biology based solution for the oral delivery of insulin. Ultimately, they came up with the ECOCO system, which is composed of E.coli Coworker and E.coli Courier. In a nutshell, E.coli Coworker is a insulin secreting gut bacteria that is designed to colonize human gut and continuously secrete a set amount of basal insulin and E.coli Courier is a non-propagating minicell that secretes insulin proportionally to the intestinal concentration of glucose.
Then, team HAFS created a pamphlet that summarises their design and headed to KDA workshop to give out the pamphlet: a biggest diabetes related workshop that provides up-to-date presentations about diabetes and gathers related medical practicioners all around Korea.
KDA workshop
Hmm...this is an interesting idea. But during the insulin therapy, the dose control is essential. Overdose or under-dose can lead to seizures and fits. I don't know how your system could rapidly reflect the necessary change of dose. Especially the E.coli coworker: how are you going to quantify the propagation of bacteria inside human gut? How can you ensure that it can actually survive to produce enough amount of Insulin. Or, the bacteria may, conversely, secrete too much insulin. That would amplify the existing side effects of insulin therapy such as weight gain. Wouldn't it be too dangerous?
Team HAFS
Hmm.. That's actually a very good point. We were also worried about the same potential hazard. We also considered working on the E.coli Courier alone. But, wouldn't that undermine the practical application of our work in terms of applying it to the currently used treatment? We would only be able to cover the part about bolus insulin.
KDA workshop
No, not at all. In fact, one of the biggest advantage of using basal and bolus insulin (4 injections) against using pre-mixed insulin analogue is the flexibility of scheduling injection. If we could only replace the three times injections to convenient capsules or tab, that would significantly improve the lives of Diabetic patients!
Taeyoon Kim
Hey, wait a minute! I think the E.coli courier can be problematic as well.
Team HAFS
What do you mean? :(
Taeyoon Kim
I am also worried about the dose control. So, you told us that the e. coli courier will secrete insulin in proportion to the "intestinal" glucose concentration, not the blood glucose level. But the intestinal glucose level cannot be a direct measure for the blood glucose level. It is rapidly absorbed after food intake. I'm not sure it would stay in the intestine that long... to make your system possible.
Team HAFS
You are right. Maybe our design simplified the entire process. We wanted to make use of the glucose concentration dependent promoter from iGEM registry.
Taeyoon Kim
What about using bile salt as a marker? Bile salt is secreted during digestion, only after the food is taken. The level of secretion depends on the amount of the food intake as well. Would this be possible? Do you guys have bile salt dependent promoter in the registry as well.
Team HAFS
Surprisingly, we do! BBa_K318514 or acRA is induced by bile salt.
Taeyoon Kim
That's great! But I have another concern. You must know that insulin is made up of two chains, and that a disulfide bond connects the two chains together. The disulfide bond is very weak and prone to denaturation. It would be better if you use a single chain insulin.
Team HAFS
Well, that's a great idea. Maybe we should try using SCI-57, a single chain insulin.
After the conversation, the project seem to have settled--a non-propagating minicell that produces insulin and lyses in response to bile salt to let out the insulin. However, there were a few more modifications of project that took afterwards when we visited NF partners to discuss about publishing patent for our oral delivery method.
Hyungsang Kim
Okay, it seems like you guys have a decent design. But one thing... isn't SCI-57 a patented molecule? In that case, you wouldn't be able to use it for your construct and get patent for it.
Team HAFS
Actually, we never thought about that. Oops, it is patented. We better find for a substitute.
Team HAFS
Oh, you know what? Last year, Team Sydney Asutralia developed an open source, unpatented single chain insulin name Winsulin! That's it, we must use that.
Lilly
Wait, I think I never heard of Winsulin. You know, a single change in base pair or amino acid sequence, or even adding fatty acid can drastically change the function of insulin analog. That's how we developed long acting and short acting insulin. You need to study more about the structure and function of the Winsulin that you guys are trying to use.
Team HAFS
Thanks, that's a valid advice. I think we should model the structure of Winsulin and its interaction with other drugs for diabetes such as metformin.
Thanks to the generous advices given by many experts, we were able to develop a more realistic design.
Our Final Project Design
Indeed, working with a oral delivery method of Insulin involves lots of factors to consider, from the digestive system, dosage control and even to the legal issues regarding patents. But ultimately, we were able to take these numerous factors into account which resulted as a insulin producing minicell that delivers bolus Winsulin, an open source single chain insulin, in response to food intake. Discover details about our design here.
