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Attributions: Kathryn Brink (Content), Shinjini Saha (Illustration)

Laboratory Safety

Over the past few months, our team worked primarily with BL1 materials (such as E. coli cultures used for cloning) and some BL2 materials (including human cell lines such as HEK293, HeLa, and Ramos). All students received training through MIT’s Environment Health and Safety (EHS) office in General Biosafety for Researchers, Managing Hazardous Waste, and General Chemical Hygiene, as well as lab-specific training. Students that worked with BL2 agents received additional blood-borne pathogens training. We also communicated regularly with Carolyn Stahl, our designated EHS representative, to ensure that we followed proper safety precautions, including meeting relevant local and national safety standards.

To keep ourselves and our samples safe, we wore proper personal protective equipment (in the form of gloves and a lab coat, as well as eyewear when necessary) at all times in the lab. Additionally, specially designated tissue culture lab coats were worn when working with mammalian cell lines, which were handled in biosafety cabinets for safety and sterility purposes. All laboratory chemicals were handled in accordance with the information on their MSDS (such as sodium azide solutions). Furthermore, nucleic acids were introduced into mammalian cells using lipofection-based transient transfections rather than viral infection, since this was determined to pose less of a safety threat.

Biological Parts

The most hazardous biological parts that we worked with during iGEM were siRNAs and plasmids encoding miRNAs against human proteins. We used siRNAs to test our miRNA profile detection mechanism. Since we were unable to find a cell line that had the same miRNA profile as that associated with Alzheimer’s disease, we transfected relevant siRNAs into mammalian (HEK293) cells to simulate the presence of the appropriate miRNAs. Whereas our miRNA project involved the use of siRNAs for testing, our approach to treatment required designing miRNA generators. We constructed miRNA generators to produce miRNAs to downregulate BACE1, an enzyme involved in the production of beta-amyloid. In order to affect expression of this endogenous protease, we decided to use a miRNA-based strategy because it could directly affect BACE1 levels in the relevant cells. Though both these siRNAs and miRNAs could have harmful health effects if they became inserted in human cells, the likelihood of this happening (especially given that proper personal protective equipment was used) is low enough so as to not pose any significant safety risks, particularly since no part of our system was ever packaged in an infectious or self-replicating vector (such as a lentivirus).

Future Implications

In addition to the physical materials that we used and the parts that we engineered, the technologies that we developed also have important safety implications. Our designed system to detect and treat Alzheimer’s disease would best serve patients if it were introduced using gene therapy, since it involves detecting endogenous miRNAs and downregulating an endogenous enzyme (BACE1). However, gene therapy techniques such as viral vector or liposome-mediated delivery can result in serious medical complications, and other mechanisms for introducing novel genetic material into cells, such as ex vivo manipulation, also carry serious risks. To date the U.S. Food and Drug Administration (FDA) Office of Cellular, Tissue and Gene Therapies (the entity responsible for regulating these kinds of treatments in the United States) has approved some cellular therapies but no gene therapy products. Given the severity of Alzheimer’s disease and the lack of suitable treatment options for this condition, however, a genetic approach like ours still has the potential to improve patient outcomes and should be considered for implementation as long as these safety concerns are taken into consideration.

Improving Biosafety with Our Synthetic B-Cell Receptor

The antibody-based sensor (synthetic B-cell receptor) that we developed to detect beta-amyloid plaques could be modified to sense other biologically relevant molecules, including those that pose a biosafety risk. By interchanging the receptor’s variable region, our sensor could be altered to detect any antigen for which there is an antibody with a known amino acid sequence. Given that our system could also be modified to produce many different types of outputs (in addition to the transcriptional activator GAL4VP16), a synthetic B-cell receptor could be of great utility for biosafety purposes and more generally as a synthetic biology tool.