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 (Bacchus, 2012)

One exciting application is in drug testing and development: tissue engineers are currently working to develop organoids (Lancaster, 2013) small tissue structures that recapitulate the behavior of organs in vitro. Organoids can be used to test drugs more rigorously in a human-like context rather than relying solely on animal models. Thus drugs can be developed with a better understanding of their toxicity and efficacy.

Organoid development promises to advance medical research and the development of clinical treatments. Tissue engineering will progress more rapidly with the development of engineered cell-to-cell communication. Engineered communication would aid in the creation of more highly networked structures and allow for better transport of factors required for differentiation (Rosello, 2010). The 2013 MIT iGEM proposes to use exosomes as a novel means of achieving engineered cell-to-cell communication.

Enabling Technology

Exosomes are 30-150nm extracellular vesicles that contain miRNAs, mature mRNAs and proteins. Discharged from sender cells via exocytosis, exosomes fuse to the target cell membrane and release their contents into the receiver cell. In 2011 Steve Gould’s lab showed that fusing GFP to the TyA protein and adding an N-terminal acylation tag led to protein oligomerization and selective targeting to the protein membrane, facilitating exosome mediated export of GFP. (Shen, 2011). 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 nuclease derived from bacteria can be directed to target specific DNA sequences by guide RNAs that are complementary to target DNA sequences. (Barrangou, 2007). We employ a mutated version of Cas9 that simply binds the target sequence rather than making an incision.

Because the Cas9/ CRISPR system can be easily manipulated to target virtually any region in the genome, Cas9 has many potential medical applications. For example, Cas9 can be used to target and modify specific DNA sequences through nuclease activity (Barrangou, 2007). As a DNA sensor, Cas9 can be used to screen cells for specific mutations. Finally, Cas9 could also be fused to transcription factors and chromatin modifiers, allowing arbitrary modulation of gene expression. Exosomes could be a means of introducing regulatory proteins to a naïve cell, opening up therapeutic avenues as well.

Our Vision

The 2013 MIT iGEM demonstrated that exosomes can be engineered to transport protein and miRNA signals of interest that can actuate a response in a receiver cell. Exosomes can be used to transport signals that are required for the differentiation and development of tissue. Two-way cell-to-cell communication will be very useful as we attempt to engineer more complex cellular networks, and the MIT iGEM teams believes that exosomal communication is an innovative means of engineering cell-to-cell communication.

Citations

Bacchus, William et al. Synthetic two-way communication between mammalian cells. Nat. Biotechnol. 30, 991–996 (2012) Lancaster, Madeline et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013) Barrangou, Rodolphe et al. CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science 314, 1709-1712 (2007) Shen, B et al. Protein targeting to exosomes/microvesicles by plasma membrane anchors. J Biol Chem. (2011)