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HP Integrated

We thought long and hard about how to present the integration of our human practice into our project. Our human practices were integral to the evolution of our project, and thus, we have decided to combine the two, and show how each facet of our human practice impacted and improved our work. Below, we present the evolution of ToloGen, from a basic concept to the complex system it is today. We’ve come a long way, and look forward to going even further in the future.

Below is a chronological map of the important milestones in our project's evolution. Further down the page you can find in-depth information regarding each milestone.

"Delay Mechanism"

After many discussions with experts, we came up with the idea of introducing a “Delay Mechanism” into our system. The delay mechanism is a crucial part of our system due to its ability to deliver our treatment without eliciting an immune response. We read about a number of controllable induction methods for mammalian cells and decided to consult an expert in order to better understand these systems and how they may fit our needs.

Meeting with Professor Ben-Zion Levi

We met with Professor Ben-Zion Levi, a researcher in the faculty of biotechnology and food engineering at the Technion. He is an expert on Molecular biology, specifically dealing with gene expression control within the immune system and its effect on pathogenic resistance and myeloid leukemia development.

Professor Levi mentioned different methods for regulating and controlling gene expression. He stated that currently there is no method in eukaryotic cells for creating an "ON/OFF" promoter. This means that any promoter we choose will “leak” to some degree.

Professor Levi suggested a few methods we could use in our project, and we discovered some exciting methods ourselves. The first method involved using an mRNA aptamer. In this method we create a loop (aptamer) in the mRNA, in such a way that if a specific ligand binds to it, it makes the loop fall and breaks the mRNA, thus limiting gene translation. Another option we investigated was the “Cumate System” which is similar to the TET system that we eventually chose. The reason we didn't choose the Cumate system is because we didn't find sufficient experimental literature, and the Cumate reagent itself was unavailable to us. Finally, after much research, and a second consultation with Professor Levi, we decided to work with the TET-OFF system. You can read more about this system HERE.

Ethics

In order to better deal with the ethical issues that we encountered we decided to reach out to an expert ethicist, Professor Laurens Landeweerd of Radboud University. After introducing our project, he raised many ethical issues concerning our treatment. We understood that to contend with these issues in an appropriate manner we needed to learn more about ethics. After attempting to delve deep into the world of ethics, we discovered that the language is very complicated, and that there were very few resources available to us as iGEMmers.

For this reason, we set out to create an “Ethics Handbook” in the hopes of learning about ethics ourselves, and creating a valuable resource for the iGEM community at the same time. In order to create this handbook, we consulted expert ethicists from around the world, read many books, and spent months writing and rewriting passages in order to make them clear and understandable to young students and non-native English speakers. With the aid of our handbook we struggled with the ethical issues concerning our project, and as a result developed a much more insightful, and meaningful, ethical outlook regarding synthetic biology. To see how these issues changed our work, please see the chapter about our project in the handbook.

Public Opinion Survey

In order to better understand the public opinion regarding our proposed treatment, we decided to conduct a survey among people suffering from allergies and autoimmune diseases. The survey was distributed throughout the country and people from various backgrounds answered it. The survey contained a questionnaire whose purpose was to examine whether or not the Israeli public is prepared to accept such an innovative and unconventional treatment. It should be noted that the respondents did not receive preliminary information about the treatment we developed prior to taking the survey. In addition, the survey was answered anonymously, by people we do not know, who aren't familiar with iGEM, or our project. You can find our survey and a summary of the answers HERE.

After analyzing the results of our survey, we learned that the public is not yet ready to fully accept and trust a procedure of this nature. In light of this realization, we decided to contend with the problem on two fronts: First, we decided that the treatment should be developed for very high risk groups who have a significant chance of developing autoimmune disease. Second, we chose to focus on the ethical and conceptual aspects of our treatment in order to help change public opinion and engender open mindedness in the general public.

In our opinion, it is merely a matter of time before the publics approach to gene therapy changes. This year (2017) the FDA approved three new treatments based on genetically engineering human cells both in-vivo and in-vitro. This lends hope that our proposed treatment may one day be acceptable to the general public.

"Kill Switch"

When presenting our project to people, many important questions came up: What would happen if a patient develops and allergic reaction to the treatment? Is the treatment reversible? What if you discover harmful side effects years later?

To contend with these issues we decided to introduce a “Kill Switch” into our system.

We read about different methods, and originally planned use the Cre-LoxP Recombination method. We read about this system and with the help of Team SMORE, we modeled the proposed system. We discovered that this system wasn’t well suited for our needs. When meeting with Professor Zila Zuckerman , we presented this dilemma to her.

Professor Zuckerman is the Director of Bone Marrow Transplantation at Rambam Hospital in Haifa. She has conducted and managed numerous clinical studies, and has a wealth of experience.

Professor Zuckerman suggested we use the Ganciclovir system. This system has already been used in gene therapies and is FDA approved. Our final design for a “Kill Switch” was based on her recommendation.

We also needed to consult Professor Zuckerman regarding HSCs and methods for harvesting them. She informed us that cord blood is rich in HSCs and easy to work with. Based on her recommendation, and ethical considerations, we developed our treatment using cord blood as the source of HSCs. We discovered that there is no danger for the baby, or the mother, in this procedure because it is done after the umbilical cord is cut ^[1].

Lastly, we needed to discuss how the HSC’s would be returned to the newborn. Bone marrow transplantation, as performed today, involves intense and dangerous conditioning routines that pose significant health risks. Obviously, such a treatment would be unthinkable for the preventative approach we were considering.

After intense research, and consulting with multiple experts, we discovered that our procedure could be performed without any prior conditioning, making it both easy and safe. This was not a simple task, as can be attested to by the conflicting opinions we received from multiple experts. Bone marrow transplants have historically been with foreign bone marrow (allogeneic transplantation), or “self” bone marrow (known as autologous transplantation) when the patient has a disease stemming from their own bone marrow. As a result, in both cases, the patient’s bone marrow must be destroyed in any case, and so, almost no research exists on autologous transplantation in healthy patients. In the end, we found multiple case studies supporting our hypothesis ^[2] ^[3] but needed an expert to verify we were correctly interpreting them. Professor Zuckerman concluded that our transplantation method is viable when working with healthy patients, and confirmed that she herself had performed such transplants successfully.

link to the regulation of public and private cord blood bank in Israel (HE)- https://www.health.gov.il/hozer/EX-003-02.pdf
Mellouli, Fethi, et al. "Bone marrow transplantation without conditioning regimen in Omenn syndrome: A case report." Pediatric transplantation 11.8 (2007): 922-926.
Endo, Morito, et al. "Syngeneic bone marrow transplantation without conditioning in a patient with paroxysmal nocturnal hemoglobinuria: in vivo evidence that the mutant stem cells have a survival advantage." Blood 88.2 (1996): 742-750.

Meeting with Dr. Ayal Hendel

Dr. Ayal Hendel is a researcher in the Institute of Nanotechnology and Advanced Materials in Bar-Ilan University, currently researching and developing CRISPR technology as a method for fixing point mutations in immune-deficient newborns.

Dr. Hendel explained the steps involved in the process of engineering CD34+ (Hematopoietic) stem cells, and answered all our questions regarding their applicability to our system.

After much deliberation, we settled on using viral transduction in our final treatment plan. Transduction is problematic because viral vectors are notoriously unpredictable and can cause significant harm if they recombine offsite. In spite of these issues, we currently plan on proceeding with an AAV (Adeno Associated Viral vector), which is notoriously safe, and has been approved by the FDA this year (2017) for use in the new and exciting drug Luxturna. We hope that with the continued development of viral vector research, they will become a more standard, and accepted therapeutic method.

After intense research, and consulting with multiple experts, we discovered that our procedure could be performed without any prior conditioning, making it both easy and safe. This was not a simple task, as can be attested to by the conflicting opinions we received from multiple experts. Bone marrow transplants have historically been with foreign bone marrow (allogeneic transplantation), or “self” bone marrow (known as autologous transplantation) when the patient has a disease stemming from their own bone marrow. As a result, in both cases, the patient’s bone marrow must be destroyed in any case, and so, almost no research exists on autologous transplantation in healthy patients. In the end, we found multiple case studies supporting our hypothesis , but needed an expert to verify we were correctly interpreting them. Professor Zuckerman concluded that our transplantation method is viable when working with healthy patients, and confirmed that she herself had performed such transplants successfully.

Manufacturing Considerations

We spoke with a representative of “Almog Diagnostics,” a supplier of "Miltenyi Biotec". We discussed our proposed therapy and currently available instruments. Most importantly, we learned about the CliniMacs Prodigy, a clinical instrument capable of automating the majority of our proposed treatment. The price of this machine is not cheap, but neither is treating these diseases.

Market Research

We investigated the market for out treatment, the costs involved in commercializing it, and the current costs of treating some of the diseases we are attempting to prevent.

Based on the American association of clinical endocrinologists, the direct and indirect medical expenses incurred treating diabetes type 1, amount to $14.4 billion annually ^[4].

In the case of Celiac disease, 1% of the population is estimated to suffer from this disease. In a paper published in 2010 - in the United States, pre-diagnostics cost of each patient was $5023 per year, after diagnosis the cost was reduced to $3295 annually ^[5].

The total amount spent treating auto immune disease and allergies annually, is over $150 billion in the US alone ^[6]. It is difficult to estimate the exact amount our treatment will cost since the technology is in constant development and the costs become significantly cheaper when creating a mass produced treatment, but as the treatment is not personalized, and can be completely handled by machines after harvesting the cord blood, we believe that one day each treatment could cost as little as $10,000. While at first this may seem like a lot of money, one must consider that the cost of vaccinating of the 4 million children born each year in the United States would only be $40 billion, a fraction of the amount currently being spent treating these diseases.

For these reasons, we believe that if and when our treatment becomes available, governments may be willing to fund the costs.

link to the website of the "american association of clinical endocrinologists" http://outpatient.aace.com/type1-diabetes/the-burden-of-type-1-diabetes
Long, K. H., et al. "The economics of coeliac disease: a population‐based study." Alimentary pharmacology & therapeutics 32.2 (2010): 261-269.‏
http://www.mdmag.com/medical-news/autoimmune-diseases-cost-us-more-than-100-billion-annually
http://www.aafa.org/page/allergy-facts.aspx

Regulations

Even though there is not enough time in iGEM to consider bringing a new drug to market, we wanted to investigate what such an endeavor would necessitate. Based on the NIH ^[7] and the Israeli Ministry of Health guide lines ^[8] , we discovered the that before any clinical trial can take place, researchers are required to bring reasonable proof that the drug may work (in our case – checking our system ,in-vivo in an animal model). Afterwards, we would have to submit detailed protocols, find a hospital to cooperate with, and more.

In an effort to delve deeper into this world, we met with Reut Ofer , CRA (clinical research associate). We presented our project to her and discussed the required approvals for clinical trials. From this conversation we understood the incredible amount of time, and money, needed in order to bring a drug to market, and the long road we face before commercialization is feasible.

When dealing with the complexities and intricacies of medical regulation, it was easy to lose sight of our final goal, and believe it impossible to bring a gene therapy to market. We were very pleased to learn that on the 30th of August 2017, Novartis, a company that has developed a gene therapy, based on genetically engineering immune cells, in order to treat childhood leukemia, received FDA approval.^[9]

This gives us hope that one day, maybe our own gene therapy will become a reality.

link to NIH - Regulations for clinical trials
https://grants.nih.gov/policy/clinical-trials/protocol-template.htm
link to the Israel Ministry of health -
https://www.health.gov.il/UnitsOffice/HD/MTI/Drugs/ClinicalTrials/Pages/CTH.aspx
link to Novartis -
https://www.novartis.com/news/media-releases/novartis-receives-first-ever-fda-approval-car-t-cell-therapy-kymriahtm-ctl019

The Future

“A smooth sea never made a skilled sailor.” Throughout our project we contended with many difficulties. With the aid of the community, experts, and industry leaders we were able to overcome many of these issues, but the road ahead is still long. We look forward to improving our project further, hand in hand with our community.