Team:MIT/Motivation

From 2013.igem.org

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Latest revision as of 03:48, 29 October 2013

Exosome Mediated Mammalian Cell-Cell Communication

Background and Enabling Technologies

The 2013 iGEM team engineered an exosome mediated cell-cell communication system. Exosomes are 30-100nm 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.

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)

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-<h1>Background and Motivation</h1>
+<h1>Background and Enabling Technologies</h1>
-<p>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.</p>
+<p>The 2013 iGEM team engineered an exosome mediated cell-cell communication system. Exosomes are 30-100nm 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. </p>
-<p>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.</p>
+<p>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.<p>
-<h1>Enabling Technology</h1>
-<p>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.</p>
+<h1>Citations</h1>
-<p>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.</p>
+<p>Bacchus, William et al. Synthetic two-way communication between mammalian cells. Nat. Biotechnol. 30, 991–996 (2012)
-<p>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.</p>
+Lancaster, Madeline et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013)
-<h1>Our Vision</h1>
+Barrangou, Rodolphe et al. CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science 314, 1709-1712 (2007)
-<p>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.</p>
+Shen, B et al. Protein targeting to exosomes/microvesicles by plasma membrane anchors. J Biol Chem. (2011)<p>
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