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From 2013.igem.org

The synthetic biology community has not come to an agreement on the precise definition of “Human Practices”. The MIT iGEM team believes there are two main aspects of “Human Practices”: Analysis of the risks, regulations, and barriers that impede the implementation of new technologies and community engagement.

MIT iGEM 2013: From Technology to Product

The team started with the goal of developing an exosome based cell-cell communication system. Because so little is known about targeting specific cargoes to exosomes, we were unsure of the feasibility of our project. Now that we have a characterized system, however, we can create a basic technological platform. In taking the first step towards creating a new product, we have chosen an application space, identified a potential market, and are interested in applying our technology to hepatocyte research and mouse model development.

1. Technological Platform

Although we focused our research on cell-cell communication, our work can be generalized. In essence, we engineered an exosome mediated protein export system. We believe this system can be useful across a range of application spaces; however, after considering the regulations we have narrowed our application space.

2. Choosing an Application Space & Considering Regulations

Because certain application spaces are tightly regulated, it can be difficult to implement a new technology. We considered three potential application spaces for our system: Research, Model Development, and Clinical Application. The exosome mediated protein export system could one day be used in a clinical setting as a therapeutic. We believe, however, that the system must first be implemented in less regulated application spaces prior to engineering and testing a clinical application. After narrowing our focused and identifying the appropriate application space for our work, we searched for a specific market that would benefit from our technology.

3. Identifying a Market

Within a broader application space there are specific markets. We needed to find a market that had a need for our technology and would be accepting of synthetic biology and exosome-based technologies. Because the biology of exosomes is not well understood, it may be difficult to market our project. While looking for a future collaborator, we found that the group most receptive to the project was already familiar with exosomes. Other groups that we contacted were not able to see an immediate application of our technology and were less interested in hearing about our work. Although marketing our technology was initially challenging, we were able to find a specific group that could use our exosome mediated protein export system as an enabling technology for basic research and mouse model development.

To bring our research into practice we first needed to choose our application space. Then we determined which systems would benefit from our exosome mediated protein export system. We found that our research can serve as an enabling technology for basic research and the creation of improved humanized mouse models. Although there are potential clinical applications, we believe it is best to target our new technology to less regulated application spaces, so we market our technology in the near future.
Figure images from Bhatia, 1998 and Chen, 2011.

Specific Application for our Technology - Potential Collaborator

To gain a better understanding of the role our research could play in the tissue engineering field, we contacted Sangeeta Bahtia’s lab at MIT. Dr. Bahtia hopes to create cell-based therapies for liver diseases and design humanized animal models for drug trials.

Hepatocyte Tissue Engineering Research: Role of Exosomes

Hepatoytes are extremely difficult to maintain in vitro. However hepatocytes that are co-cultured with mouse fibroblast cells can successfully survive in culture (Bhatia, 1998). Knockdown of an exosome secretion regulator in the mouse fibroblast cells, leaves the mouse cells incapable of supporting the hepatocytes.

Therefore, some factor in mouse cell exosomes helps support hepatocytes in vitro.

Impact: Our Work as an Enabling Technology to Hepatocyte Research

We believe out exosome mediated protein export system can serve as an enabling technology for advancing research on the role exosomes play in hepatocyte survival in vitro. As the Bahtia lab gains more information about the RNA and protein factors that allow mouse exosomes to support hepatocytes, we can engineer sender cells to specifically export factors of interest. Our engineered exomoses can be used to determine which proteins are necessary to support the hepatocytes. Ultimately exosomes produced by our engineered sender cells could allow tissue engineers to culture hepatocytes without the addition of mouse fibroblast cells.

Impact: Our Work and the Development of Better Mouse Models

Considering the role the liver plays in human drug metabolism, animal models that exhibit humanized liver function are extremely important for drug testing. In 2011 the Bahtia lab created human ectopic engineered artificial liver (HEAL), by co-culturing hepatocytes and fibroblast cells that were then encapsulated with liver endothelial cells. The humanized liver was then transplanted into mice where they recapitulate numerous human liver functions. The humanized mice provide a model for drug testing (Chen, 2011). This model, however, could be improved if the artificial liver was made solely of human cells and did not need the mouse support cells. If we could treat the hepatocytes with exosomes containing the necessary protein or RNA factors, hepatocytes could be cultured without mouse fibroblasts and used to create artificial livers that mirror human liver activity more closely.

Requirements for Our System

Identifying our application space and gaining a better understanding of our market has helped to guide our future work. We have come to realize that our system must have three main components to be most useful:

1. We must be able to specifically target cargoes of interest to an exosome. We have demonstrated that we can specifically targeted proteins to exosomes; however, more research must be done to find a means of targeting RNA to an exosome.
2. Our exosomes should be able to target specific cell types. Perhaps this can be achieved by modifying cell surface receptors on the exosomes, so only the desired cell types take up the vesicles.
3. Our exosomes carrying the cargo of interest would be even more useful if they were detectable. This would allow users of our system to easily verify the presence of exosomes when they are added to an in vivo system, like a mouse model, or and in vitro system, like a plate of cultured cells. Because exosomes are so small, detectability is difficult to accomplish and is an issue we hope to focus on in the future.

By considering human practices we have identified a potential collaborator and gained an understanding of the needs of our target market.

MIT iGEM 2013 Engagement Efforts

We participated in a series of engagement exercises, educating our international collaborators to promote the formation of a new iGEM team, risk assessment of our project with Kenneth Oye, consulting with patients and doctors about potential application spaces for our research technology, educational outreach to the local Boston community, and collaborating with fellow iGEM teams to improve their projects and presentations.

International Education

In January of this year, we ran a student-run laboratory course at MIT for 2 weeks over IAP http://stellar.mit.edu/S/project/synbio-iap2011/. IAP is a special 4 week term in January, over which students can take classes and participate in a wide variety of classes. The curriculum we teach is half seminar and half in a biology lab, exposing neophyte researchers to the origins of synthetic biology and current best practices. We include lectures on iGEM, the parts registry, the use of abstraction, and how theory and experiments can be complementary. These lectures are followed with an intensive lab setting where teams of students utilize BioBricks to build 2 plasmids, forming an inducible bioluminescent circuit. This year, we doubled enrollment by co-teaching the class with a dozen students at the Universidad Adolfo Ibáñez in Santiago, Chile. We streamed our lectures to South America and additionally dispatched three MIT iGEM students to Santiago to run the lab sections. This international engagement will be fully realized for the 2014 competition year when UAI will host their own iGEM team for the first time – one that we will assist as well as have applied for funds to allow an exchange program for the two teams. In July of this year, the professor and lead instructor from Chile that will be forming the new team came to Boston to shadow the iGEM team for several days to learn and develop good habits of team management.

Safety and Risk Assessment

In addition to our international involvement, we also pursued several projects locally in the Boston area. The first was hosting Kenneth Oye http://web.mit.edu/polisci/people/faculty/kenneth-oye.html for an hour long seminar to the team on a discussion on the implications of our project ideas. During the course of our conversation with Professor Oye, we first went through several case studies in biological research before turning our attention in working to analyze our own project for risk, safety, legal, or ethical concerns.

Mammalian cells can be engineered through a variety of methods – the use of viral vectors that integrate into the genome, exogenous introduction of proteins or other factors, or transient transfection of non-replicating DNA. The use of viral vectors, while common in laboratory settings, would be a certain critical risk in containment and safety of our undergrad researchers due to the ability of some classes of viruses to infect humans in addition to test tubes of cells. The use of proteins or external cues did not afford the possibility to manipulate our mammalian cells in the necessary fashion and thus we chose to only use transient transfection. Because transient transfection does not use replicating DNA, it represents a negligible risk to users when decontaminated and disposed according to MIT Environmental Health and Safety regulations https://ehs.mit.edu/site/. We used similar risk matrices to arrive on decisions of what cell lines to engineer, the use of various reagents in our work, and the policy on necessary personal protective equipment during experiments.

Outreach to the Non-Technical Community

As part of an ongoing thrust to introduce synthetic biology to the community at large outside of research universities, we pursued a partnership with the Museum of Science in Boston. A teammate’s friend was an intern at the Museum and we were able to secure a meeting between one of the outreach coordinators, Miriam Ledley, and our team on introducing a display into the Theater of Electricity on applying circuit design into the biological sciences. Miriam lead us through the specifications required of an exhibit – the educational content required, the build quality of an apparatus, the age-group of participants, and procedural aspects for biocontainment. For instance, initial ideas involved students streaking out rapidly dividing fluorescent bacteria on solid agar plates and visualizing their bacteria under a black light in the afternoon as well as assembling circuits using physical block abstractions. While we were able to design a unified lesson plan for inclusion in the exhibit, the balance between engaging children with biological material in a safe manner has become a much harder than anticipated challenge. The lack of a contained lab environment in the museum as well as the infrastructure necessary to house bacterial stocks, plates, and project supplies were above and beyond what our team could provide to the museum. We have since followed up with the MIT Center for Environmental Health and Safety (CEHS) to build off some of their experience using Lego DNA Kits in the Cambridge Science Festival and apply a similar LEGO model approach to synthetic biology as an activity. Tentatively, we are slated to assemble and deliver our first prototype kit for feedback from users in the Synthetic Biology Center in mid-November of this year.

Collaboration within the Academic and iGEM Community

In ambitious research projects, an extreme amount of collaboration must occur between far-flung research labs. This year, we had the opportunity to encounter several key research experiences during our project. The first was securing sequence and physical DNA in published literature from the originating lab. For multiple constructs, the lab’s principle investigators did not respond to DNA requests and made it very difficult for us to tackle our project. Thankfully, we were able to reverse engineer some constructs from existing literature methods sections as well as find suitable replacements from commercial or other academic sources. Speaking to researchers within the Synthetic Biology Center at MIT, this lapse in communication appears to be chronic in academia and illustrated to us the importance of standardized DNA repositories. For iGEM, the registry’s inclusion of both working and planning parts are instrumental in being able to push iGEM projects forward while other common repositories such as AddGene or NCBI GenBank serve the same purpose for the larger scientific community. The second challenge we faced was being ‘scooped’ this summer by a larger research lab on an idea we had conceived as part of our iGEM project. Throughout the course of the summer, we had elected to investigate the Cas9-CRISPR system’s use as a communicating message in exosomes and built a transcriptional activating version of Cas9, Cas9-VP16 https://2013.igem.org/Team:MIT/Cas9-VP16. However, several weeks into our iGEM project as we were testing our Cas9-VP16, a manuscript was published that effectively scooped our project with the protein Cas9-VP64, a transcriptional activator of similar design to our proposed construct http://www.nature.com/nbt/journal/v31/n9/full/nbt.2675.html. Thankfully, we were able to utilize the work in the manuscript to better our design but it was still an eye-opening experience that in future research endeavors we need to be aware about.

Finally, we worked to help our fellow Boston area teams in two avenues. In the first, we worked with Wellesley on testing their software programs to provide valuable feedback from an experimental background on their tools to help experimentalists https://2013.igem.org/Team:Wellesley_Desyne/Human_Practices. We also took them on tours of our facilities to enable them to get an understanding of our workflow from design -> data to allow them to further brainstorm on effective tool development. In the second Boston area engagement, we participated in NEGEM hosted by Boston University’s iGEM team. NEGEM is a gathering of local New England teams to give practice talks about their iGEM projects and garner feedback while building camaraderie.

References

• Chen, A et. al. Humanized mice with ectopic artificial liver. Proc Natl Acad Sci U S A, 108: 11842-11847. (2011)
• Bhatia, S et. al. Microfabrication of Hepatocyte/Fibroblast Co-cultures: Role of Homotypic Cell Interactions. Biotechnol. Prog. 14, 378−387. (1998)