Vitamin A Deficiency and the Creation of Oviita
Vitamin A deficiency (VAD) has long been a problem in vulnerable and developing areas. The topic has been drastically underreported by media and major organizations since the early 2000s (Baker, 2019), and therefore often goes overlooked. However, it turns out that vitamin A deficiency cases have reached a seven-year high, and numbers are expected to rise (Baker, 2019). Providing a solution to vitamin A deficiency would not only prevent hundreds of thousands of cases of childhood blindness a year (WHO, 2013), but also improve immunity to other diseases. Oviita’s vision is to see no child blinded by vitamin A deficiency. For this to happen, we wanted to make informed decisions. After all, many organizations have invested millions of dollars to alleviate this problem.
To fully understand the problem and design a plan to address it, our team utilized iGEM Calgary’s human-centered design process which leads to integration with key stakeholders and end users throughout the project lifecycle. To do this, with each new idea, we cycled iteratively through the HP pathway:
- UNDERSTANDING THE PROBLEM
- DESIGN SOLUTIONS
- EVALUATE AND ITERATE
Initially we thought that supplementing beta-carotene alone would solve the problem. Little did we know that vitamin A deficiency was caused by multiple gaps/problems with compounding effects. This page documents the story of Oviita from understanding vitamin A deficiency, to ideation, and implementation of each subproject addressing bottlenecks in the vitamin A supplementation pipeline.
UNDERSTANDING THE PROBLEM
Before investing our energies in doing wet lab and dry lab work, we wanted to verify that vitamin A deficiency is still a problem. We talked to people who work directly with vitamin A deficiency: nutritionists, public health specialists, anthropologists, and doctors. We created conversation about the problems with past attempts in vitamin A supplementation programs, what the supplementation process is like, the current status of vitamin A internationally and countries they are in, and the bottlenecks in the supplementation process.
What did we discover?Current State of Vitamin A Deficiency
Our HP contacts who work in the Philippines, Ghana, India, and Burkina Faso told us that VAD still affects a significant percentage of the children in their countries. The government plays a big role in promotion of growing beta-carotene biofortified crops and supplying vitamin A (retinol) supplement capsules. However, these efforts are not enough.
Lack of Sustainable Solutions
Many institutions from western companies have provided VAD-vulnerable countries with resources to combat the problem, but we learned that they don’t work well because these institutions do not leave infrastructure for the people to work with. As a result, their efforts go in vain as soon as they leave the country and stop providing resources. Many of these efforts also do not consider the culture and available resources in the country they are helping. Lastly, the money has to come from someone’s pocket. Even with cheap vitamin A capsule prices, transportation and distribution costs can still be economically straining. Most importantly, the people we are working with do not want external aid enforced on them. They want something sustainable-- something THEY can have ownership and accountability of.
Minimal Community-based Implementation
Past approaches such as the Golden Rice project and biofortification of other staple crops through genetic engineering did not work well because plants need months to grow and farmers would suffer economic losses because the biofortified crops can contaminate their non-fortified crops and prevent its export. Additionally, vitamin A supplementation through gel capsules is only done biannually. Health and nutritional organizations allot for more than 100% coverage of supplements, but social workers and mobile nurses are not always able to reach rural areas.
Inadequate Access to Healthcare
Vitamin A supplementation is not always effective because children's intestines are often perforated by worms and parasites due to lack of proper footwear and access to clean water in these areas. As a result, deworming using Albendazole is coupled with the vitamin’s supplementation. Asides from these initiatives, we were surprised to hear that diagnostic methods for VAD are based on height, sight, and academic performance tests rather than laboratory testing. This explains the sparse data on Vitamin A deficiency by the WHO, which has not been updated internationally since 2005. From Dr. Sanou’s Public Health perspective, these tests are inaccurate and are not a good representative of the actual numbers on vitamin A deficiency.
Having identified key problems in the vitamin A supplementation distribution, our team started brainstorming ideas. Upon careful consideration of our stakeholders’ needs, we decided on using a chassis producing β-carotene targeted for consumption, like nutritional yeast. Upon conducting literature review, we decided on working with Rhodotorula toruloides, a red oleaginous yeast which naturally produces β-carotene. Then, we would genetically modify it with a series of cellulases to use agricultural waste as a feedstock. In our first plan, for added nutritional benefits, we would engineer two additional genes to our chassis for expression of EPA, an omega-3 fatty acid known to have cognitive benefits. A light-inducible kill switch would also be added as a biocontainment measure. For added safety features and optimized growth conditions, our yeast would be grown in a laboratory bioreactor. With these attributes, the yeast could be grown in a community center, clinics, or dispensaries-- improving vitamin coverage and delivery in a sustainable manner.
To learn more about these solutions, please click the buttons below.
With a proposal on hand, we talked to our end users to verify its need and discuss its implementation in their workplace and communities. We talked to healthcare workers and nutritionists in rural areas in South Asia, Sub-Saharan Africa, and Southeast Asia which are among the most VAD-vulnerable areas.
The feedback we received from our stakeholders was that a bioreactor capable of producing a steady supply of vitamin-A producing yeast would be much better than waiting for biannual distribution of the vitamin. Omega-3 fatty acids are an emerging essential nutrient with numerous health benefits which make its production favourable.
Dr. Sanou mentioned that even with omega-3 supplementation, intestinal parasites will still pose a problem in preventing the absorption of not only vitamin A but the omega-3 fatty acids too. Because of this, our team decided to change the production of omega-3 to that of thymol, an anthelmintic compound found in thyme leaves. The compound is shown to critically damage Caenorhabditis elegans and its eggs, which is the gold standard in anthelmintic drug testing. While this is not a cure to intestinal parasites, it can mitigate intestinal damage and improve general micronutrient absorption until drugs such as Albendazole are distributed, even without access to clean water or proper footwear.
In places like the Philippines, India, Burkina Faso, and Uganda, people would have events like “nutrition week” or “micronutrient week” dedicated to diagnosing VAD, deworming using Albendazole, supplying vitamin A, and also informing parents and children about the importance of incorporating vitamin A in the diet. Dharamwati thinks that having a communal FAB bioreactor would be better than individual ones for homes as it would be built with shared investment, and encourage community participation especially in events such as nutrition week.
We talked to Tyler Warnock, a medical school student who has worked with Healthy Child Uganda and Mama na Mtoto in Tanzania about his experience with global health work in Uganda, and how we could apply them towards our bioreactor and project implementation. He cautioned that making a difference is all about relationship building, and its absence would make our project implementation very difficult and “ unethical”. Many western people come in to collect data and leave without infrastructure, causing a divide between them and the local people.
With unethical implementation and inaccurate testing in mind, our team worked on the Randle Cell Testing Device which detects vitamin A levels in the blood. This is important because it helps us build a more accurate picture of VAD in our target areas, and informs us and our end-users whether our solution is making a difference.
To learn more about the Randle Cell Testing Device and Thymol Production, please click the buttons below.
We also talked to Dr. Samir Gupta, one of the co-founders of the Health Program at the Global Pathway School in India, which was developed by Canadians to help Indians students living below the poverty-line. We learned about their application of a 15-point plan which includes feeding the students meals in school, and supplying vitamin A and Albendazole for deworming. We asked him how he and his team successfully built this community without being culturally insensitive. He said that it is important to employ local people in leadership roles and in positions which enable community members to connect with them around economic and social problems. For the implementation of our yeast, he suggested considering schools for distribution since kids would be in one location being fed.
Our HP contacts agree that having microenterprises linked to Oviita would make our project more self-sustaining as it would train the local people while providing empowerment and employment, especially to women. Taking Dr. Gupta’s feedback in consideration, we partnered with the West Nile Youth Empowerment Centre (WNYEC) in Uganda which empowers youth through cultural and entrepreneurial training across eight different regions in Uganda. After explaining our project and its microenterprise opportunities, Nadia Iddi, the programs officer of WNYEC, said that this would be a great idea as it would create employment opportunities in their area. As long as our team provided them the initial training, they would also train their community members in how to operate our bioreactor, creating products like yoghurt fortified with Oviita, and educate people with the importance of vitamin A in the diet. Once the proposed training model is set in place, our partnership would expand to nearby health clinics around the area. Nadia informed us that the general perception for genetically modified organisms in Uganda is negative, but that should not be a threat with its monetary and health benefits. Over the next year, we will be working closely with them to see how we could best integrate the Oviita system tailored to their resources and culture.
The graphic above represents the Oviita system we've build hand-in-hand (virtually) with our end-users after various iterations. Now that we had a stakeholder-approved proposal, it was time to figure out how to make the science work.
Expert ConsultationsCELLULASE ENGINEERING
Vitamin A supplements are typically provided or sold by foreign bodies. If the supply-chain are disrupted, VAD-struck countries would not receive the vitamin. Unfortunately, this is the case with many humanitarian organizations such as Save The Children International, which halted supplement and material donations to vulnerable countries due to COVID-19 (Personal Communications, Save the Children US). Thus, we needed to come up with a self-sustaining solution that the community could maintain. Many of these communities grow crops for a living, so we thought to engineer cellulases into our yeast. Since our team has no experience working with Yarrowia lipolytica, we needed to know what considerations were required for our system.
Vitamin A supplementation is coupled with deworming with compounds like Albendazole twice a year. This is because intestinal parasites perforate the intestines and prevent the maximal absorption of vitamin A and other micronutrients. Despite biannual deworming, lack of access to clean water and proper footwear only causes intestinal parasites to return. Additionally, these worms may also develop drug resistance especially since anthelmintic and deworming agents commercially available are limited. To alleviate intestinal damage, we decided to genetically engineer our yeast to produce thymol, a compound found in thyme leaves. Thymol has been shown to have anthelmintic properties against Caenorhabditis elegans and their eggs.
Our team had no clue how anthelmintics worked, or how drugs like Albendazole transition from the laboratory to schools. To find out more, we talked to a molecular and developmental geneticist, a doctor, and a parasitologist.
A synthetic biology project without a biocontainment strategy would simply be irresponsible. If we wanted our yeast to be deployed outside a lab setting and grown in community FAB bioreactors, we needed to observe extra precautions, making sure we don't do ecological damage to nature. We initially played around with the idea of having a light-inducible kill-switch system, but we weren't sure if that was sufficient.
Thus, we talked to a series of biochemists and molecular biologists who knew a thing or two about effective biocontainment strategies, before finally deciding on an auxotrophic co-culture system.
Growing conditions for our yeast must be regulated so we can maintain sufficient beta-carotene production and control beta-carotene doses for consumption. Proper design is also needed to make sure our yeast is contained in a closed-system, preventing accidental ecological damage.
RANDLE CELL TESTING DEVICE
Available data on VAD is obsolete, dating back to 2005. This data was not obtained through testing of retinol concentrations in the blood, but through a series of diagnostic tests scrutinizing vision, height, and academic performance. For Oviita to really make a difference, we need to know whether our supplementation project is needed by our end-users. This is why we created the Randle's Cell Testing Device, a field-based test which utilizes aptamers to quantify retinol binding protein concentrations in the blood. These concentrations can then be correlated to Vitamin A concentrations, enabling communities to detect VAD effectively.
We reached out to professionals from various engineering principles to help the device come to fruition.
Evaluate & Iterate on Design
Further Improving Our DesignFACULTY TALK
Shortly after forming our initial proposal, we assembled stakeholders ranging from nutritional anthropologists to electrochemists to evaluate our project proposal. Overall, the feedback we received was promising. However, many questions were raised towards the effectiveness of our light-inducible kill switch, our choice of chassis (Rhodotorula toruloides) and its safety status for consumption, and how we would assemble our constructs for engineering. We were informed that most kill switch systems are susceptible to mutations as there is little reason for the cell to keep the trait. Our system would also have problems if our yeast reaches open waters or goes deep into the soil where light levels vary from minimal to none. We were advised to explore auxotrophy or quorum sensing as an alternative biocontainment strategy. Many of our guests have not heard of our chassis before, and expressed concerns about our ability to engineer it, let alone consume it. We were advised to consider this thoroughly, and use a food-safe yeast such as baker’s yeast instead. While we did not use baker’s yeast, we made the transition to Yarrowia lipolytica, a Generally Recognized As Safe (GRAS)-designated yeast which is oleaginous. Having an oleaginous yeast would aid in vitamin A delivery as beta carotene and retinol are lipophilic. The strain we graciously received from Dr. Ledesma-Amaro’s laboratory in London has been engineered to produce the carotenoid already. Lastly, our attendees educated us about the wonders of Gibson Assembly and how it would save us a lot of trouble while assembling the constructs we needed.Julygem
We hosted a two-day online meetup in July, where teams from Canada, USA, Taiwan, and Mexico got the chance to share their projects and receive feedback from experienced judges, as well as hear a number of talks by experts in the field that followed the overall theme of translating synthetic biology into the real world. Teams were asked to prepare a 5-7 minute pitch on their project to be presented in front of judges that were not all experienced biologists. Teams that evaluated our pitch though that we should talk to more stakeholders and integrate their needs and feedback to our presentation. The teams also thought we could do a better job of explaining how our solution provides a competitive edge towards existing solutions in vitamin A supplementation, and how both wet lab and dry lab are working together to address the problem more clearly.iGEM Concordia's Mini Jamboree
We were fortunate to be invited by iGEM Concordia to participate in the Mini Jamboree where we had a friendly competition with four other Canadian teams. The judges thought we were on the right track on working with the local taste palettes and engineering principles. We were cautioned to check our assumptions about the societies we were working with in terms of building materials. Our audience was a bit lost with the values for vitamin A and EPA and how they are linked together, and thought a model on our co-culture switch system would be worth considering. The other teams also agreed that we needed to provide more reasoning why our project was important.cGEM
We participated at cGEM, a Canada-wide iGEM competition where we presented a poster and a 20-minute project presentation. The area where our judges and fellow teams thought we were lacking the most was showing the integration of our stakeholders throughout the process in our presentation, and whether we have talked to our end-users in vitamin-A deficient areas. At the time, we did not talk to local communities and public health workers so we made sure to do that and reached out to people in India, Philippines, and Uganda. In particular, they were concerned about having a training program or system set in place to ensure the communities knew how our bioreactor would operate, and how the yeast would be properly incorporated into their diets. We were also advised to address any possible obstacles we might face with our solutions
Improving Oviita Beyond the 2020 JamboreeWORKING WITH WEST NILE YOUTH EMPOWERMENT CENTER IN UGANDA
The West Nile Youth Empowerment Center Uganda is an NGO which empowers youth through cultural and entrepreneurial training across eight different regions in Uganda. This partnership is crucial because we would like to see Oviita’s implementation come to fruition. Dr. Gupta emphasized the importance of having the local people take over leadership roles for Oviita to be successful and self-sustaining. Oviita’s pilot program with the WNYEC is important as other implementations may arise. We will be working closely with them and providing training in the bioreactor’s operation. In exchange, we will also receive training on working with their community and others they are working with. We will be observing models Dr. Sanou recommended, where people get trained in women’s centers on micro entrepreneurial ventures and determine whether our system is working for this environment.
We recently connected with Western Heads East which is a collaboration between Western University staff, students, faculty and African partners using probiotic foods to contribute to health and sustainable development. We talked to Robert Gough, who was more than willing to partner with us and provide information, contacts and resources. He stressed partnerships are huge. Especially on the ground ones and with local universities. He considers public perception of GMOs to be the most challenging aspect of our project. His advice on implementation is to first reach out to a university in Ghana, start a research program to include things like bioreactor, social enterprises and general implementation. During this time, we would also be working through the local regulations. For ethical reasons, any research we would hope to gain from this should be in tangent with local groups. Next we were advised to contact a local organization or hire a local director of operations and then start a specific flagship pilot program. Afterwhich, we would build one bioreactor and then monitor all the data from it and its impact on the community. For Western Heads East, it took four years before they expanded from one kitchen to multiple areas. After sufficient data is collected, we can then use that to market our idea for expansion. For more details, see Entrepreneurship.
Creating an Industrial-Grade Bioreactor with BIONET
As the wiki freezes, BIONET, A Spanish company that designs and supplies bioreactors, reached out to us to provide our team with their support and technologies. Our team will be working on the development phases and equipment requirements in more detail to get this collaboration started after the end of the 2020 Jamboree.
Baker, S. (2019, July 1). The danger of complacency: Lost progress in vitamin A distribution. Retrieved October 28, 2020, from https://ww2.gatesfoundation.org/ideas/articles/lost-progress-in-vitamin-a-distribution
World Health Organization (WHO). (2013). Micronutrient deficiencies. World Health Organization (WHO). https://www.who.int/nutrition/topics/vad/en/#:%7E:text=Vitamin%20A%20deficiency%20(VAD)%20is,the%20risk%20of%20maternal%20mortality.