From 2013.igem.org

Background and Motivation

This summer, the 2013 MIT iGEM team worked to engineer exosome mediated cell-to-cell communication. In vivo cell-to-cell communication is vital for pattern formation, organ development, coordinated responses to environmental changes, and the maintenance of an organism. Engineered cell-to-cell communication would help rapidly advance the field of tissue engineering.

In America alone, over 100,000 people are in need of an organ donation, and the supply cannot meet the demand. Moreover, it is often difficult to find a compatible donor, and transplant rejection is not uncommon. Engineered cell-to-cell communication could program cells from a patient to assemble tissues of interest. The ability to engineer the assembly multi-cellular structures could eventually lead to the construction of synthetic organs that can be transplanted into patients. Synthetic tissues can also be used for drug development. By creating small synthetic tissues, organoids, that mimic the in vivo drug response, we can rigorously test new drugs in order to determine efficacy and toxicity. Engineered mammalian cell-to-cell communication could aid in the creation of new therapies and tissue engineering strategies.

Enabling Technology

We propose the use of exosomes to achieve engineered cell-to-cell communication. Exosomes are 30-150nm extracellular vesicles that contain miRNA, mRNA and protein. Discharged from sender cells via exocytosis, exosomes fuse to the target cell membrane releasing their contents into the receiver cell. Despite their small size, exosomes can be isolated through ultracentrifugation or the use of the Invirogen’s Total Exosome Isolation Reagent. Isolated exosomes still contain intact miRNA and protein that can be used to actuate a response in receiver cells.

In 2011 Steve Gould’s lab (John’s Hopkins School of Medicine) showed that fusing GFP to Acyl-Tya lead exosome mediated export of GFP. Acyl-Tya causes oligermization and selective targeting of the desired protein to the membrane where it can be packaged into an exosome. We hope to engineer sender cells that export proteins of interest, by fusing them to Acyl-Tya.

Our project also utilizes the Cas9/CRISPR system. Cas9, a DNA binding protein, can be directed to target specific DNA sequences. The Cas9/ CRISPR system functions as the prokaryotic immune system. The prokaryote creates guide RNAs that are complementary to foreign viral DNA sequences. The guide RNA is loaded into a Cas9 nuclease. Once Cas9 has found the target sequence, it can excise it from the genome. We employ a mutated version of Cas9 that simply binds the target sequence rather than making an incision. We use the Cas9/CRISPR system to create a transcriptional activator and novel DNA sensor. We hope to develop novel systems that utilize Cas9 and package them into exosomes. Exosomes will provide a means of delivering Cas9 constructs that could have numerous medical applications.

Our Vision

We hope to engineer cell-to-cell communication by utilizing miRNA and protein signals. Certain miRNAs, like mir503 and mir451, are selectively targeted to exosomes. Thus we design receiver circuits that respond to mir503 and mir45 in order to show that exosomes can be used to actuate receiver cell circuits. We also constructed a variety of protein signals including transcriptional activators, translational repressors, DNA binding proteins, and DNA recombinases. We believe that exosome mediated cell-to-cell communication can one day be used to influence to behavior of unengineered cells, which could have interesting tissue engineering applications.