Difference between revisions of "Team:BGU Israel/Description"

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         <br /><br />
 
         <br /><br />
  
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<p>Tell us about your project, describe what moves you and why this is something important for your team.</p>
 
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<br />
 
  
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<h5>What should this page contain?</h5>
 
 
<ul>
 
<ul>
  
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<h4> Overview </h4>
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<h2> Overview </h2><br />
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<h3><i>Motivation</i></h3><br />
 
<p>
 
<p>
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This summer we have set our goal to design and test a synthetic machine which could distinguish individual cancer cells from healthy tissue, and that works only when inside the nucleus of a human cancer cells. We have constructed two separate designs: the first, for reducing tumor proliferation and inducing apoptosis by knock-down of vital genes. The second is for expression of exogenous proteins which could color the tumor in a visible way for the naked eye (for example, a chromophore), or lead to cell apoptosis by expression of an apoptotic protein.
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Although it is one of the most researched and funded fields in medicine, cancer is still a major cause of morbidity and mortality worldwide, with 14 million new cases and over 8 million deaths per year.
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The first design utilizes CRISPR-cas9 and gRNA to knock-down a vital gene, namely, Ubb gene which encodes for poli-Ubiquitin. Ubiquiting levels are elevated in most, if not all human cancer cells, are essential to the growth of cancer cells and the protein is thought to help cancer cells adapt to increased stress. Ubb downregulation by si-RNA has shown a great decrease in tumor proliferation and more than 50 percent increase in apoptotic cells (1).
+
It is the second cause of death worldwide, and it’s responsible for quarter of the death cases among developed countries. If current trends continue, cancer will soon surpass heart disease as the leading cause of death in the U.S
 +
<br />
 +
The failure of current therapies to cure cancer is due to a few reasons:<br />
 +
1. Most treatments cannot distinguish precisely enough between cancer and healthy cells. Low specificity means higher toxicity and high rate of adverse effects.<br /><br />
 +
2. Cancer cells have an extremely complex pathophysiology with multiple biological pathways allowing their infinite growth and resistance to treatment. Thus, intervening with only one of this pathways, as most current therapies do, is doomed to fail.<br /><br />
 +
3. Cancer is not a single disease, but a collection of diseases arising from different genetic mutations, involving abnormal cell growth.
 +
<br /><br />
 +
 
 +
<b> Our aim </b>, therefore, is to develop the ideal cancer therapy that is both highly specific for cancer cells, efficient, and personalized for each tumor and patient genetics. <br />
 +
</ p>
 +
 
 +
<h3><i>Boomerang</i></h3><br />
 +
 
 +
<p>
 +
This summer we have set our goal to design and test a synthetic machine which could distinguish individual cancer cells from healthy tissue. Our design makes sure that the function of our machine will be limited exclusively to cancer cells. Our machine does so by being operated by 2 separate cancer-specific promoters, which are highly and predominantly activated in cancer cells (1)+ <a href="">(link to Results figure of TERT and survivin)</a>.  
 +
By using two separate promoters we ensure that our system will be exclusively activated only in cancer cells, with minimal, if any, expression in healthy cells. Simply by changing the promoters that control the system parts, our modular system makes it easy to design the system to fit the genetic profile of each individual malignancy. <br />
 +
<br />
 +
 
 +
There were several ways in which we can deliver our system in the body, and we chose AAV (Adeno Associated Virus) because of its many advantages, including low pathogenicity and mild immune response. AAV is used today in advanced clinical trials for gene therapy. The efficacy of our system will be dependent on the development of effective delivery approaches. (3).
 +
<br /><br />
 +
 
 +
In our specific design for the prototype/proof-of-concept studies we use promoters which are linked to tumor proliferation (human telomerase-reverse transcriptase (hTERT) promoter) and enhanced survival (human survivin promoter), both known to be highly active in multiple cancer cell types.  
 
</p>
 
</p>
  
 
<h4> The Design </h4>
 
<h4> The Design </h4>
 
<p>
 
<p>
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The design includes two parts: one with cas9 gene, and the other with a gRNA compatible to three sequences in the second axon of Ubb.
+
We have constructed two separate designs, both utilizing different versions of CRISPR/Cas9 system: <br />
−
Cas9 – the cas9 endonuclease was put under control of h-tert promoter, thus should be expressed predominantly in cells in which it is highly active, namely – cancer cells. When guided with gRNA, cas9 cuts both strands of DNA at the target sites, which leads to the cell trying to fix the double strand break, adding mutations which damage the activity of the mature protein.
+
<ul>
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gRNA – the guide RNA is a hundred bases long molecule with a unique two dimensional structure which binds cas9 and guides it to a dsDNA sequence complementary to 22 base pairs on the 5' end of the molecule.
+
  <li> Knock-out of genes essential for cancer cell survival (e.g., to inhibit tumor proliferation and induce apoptosis) </li>
 +
  <li>Expression of exogenous proteins which could: 1) label the tumor in a way which would enable surgeons to identify its edges for its complete removal (e.g., a chromophore), 2) lead to cancer cell death (e.g., by expression of an apoptotic protein); and 3) to produce a biomarker detectable in blood and/or urine for cancer diagnosis</li>
 +
</ul>
 
</p>
 
</p>
  
−
The second design includes the gRNA and two different parts.
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<table>
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dCas9-V64 – dCas9-V64 was engineered so that it lacks endonuclease activity and has 4 V16 activation domains attached. When guided to a specific promoter, dCas9-V64 promotes transcription of genes downstream of binding site.
+
  <tr>
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The third part is synthetic promoter regulating GFP. The synthetic promoter has 3 complementary sites for the gRNA from the second part, which, upon binding, should paint cancer cells fluorescent green.
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    <th> <img src="https://static.igem.org/mediawiki/2015/archive/7/72/20150903083258%21BGUigem_project_design1.png" alt="Smiley face" height="310" width="473"></th>
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**This construct was made as a proof of concept. By utilizing a synthetic promoter we could, in theory, express an apoptotic protein, a toxin and pretty much everything with a promoter.
+
    <th>TBD</th>
 +
  </tr>
 +
</table>
 +
 
  
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<li>A detailed explanation of why your team chose to work on this particular project.</li>
 
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Cancer is the number one cause of mortality in developed countries
 
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<li>References and sources to document your research.</li>
 
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<li>Use illustrations and other visual resources to explain your project.</li>
 
 
</ul>
 
</ul>
  
  
 
<br />
 
<br />
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<h4>Advice on writing your Project Description</h4>
 
  
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<p>
+
<h4>Why Boomerang?</h4>
−
We encourage you to put up a lot of information and content on your wiki, but we also encourage you to include summaries as much as possible. If you think of the sections in your project description as the sections in a publication, you should try to be consist, accurate and unambiguous in your achievements.
+
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</p>
+
  
 
<p>
 
<p>
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Judges like to read your wiki and know exactly what you have achieved. This is how you should think about these sections; from the point of view of the judge evaluating you at the end of the year.
+
<table>
 +
  <tr>
 +
    <th> <img src="https://static.igem.org/mediawiki/2015/8/83/Igem_bgu_logo.png" height="60px" width="245px"></th>
 +
    <th>Like a boomerang (boomerang logo) thrown by a person which flies back instantly, our synthetic machine uses cancer cells' own genetic alterations against them.</th>
 +
  </tr>
 +
</table>
 
</p>
 
</p>
 +
  
  
 
<br />
 
<br />
 
<h4>References</h4>
 
<h4>References</h4>
−
<p> (<a href="http://www.nature.com/srep/2013/130911/srep02623/full/srep02623.html#">1</a>) Downregulation of ubiquitin level via
+
<p> (<a href="http://www.nature.com/gt/journal/v8/n7/pdf/3301421a.pdf" target="_blank">1</a>) The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters</p>
−
knockdown of polyubiquitin gene Ubb as
+
−
potential cancer therapeutic intervention
+
−
Choongseob Oh
+
−
, Soonyong Park
+
−
, Eun Kyung Lee2 & Yung Joon Yoo </p>
+
 
    
 
    
  
−
 
+
<p> (<a href="http://www.nature.com/nrc/journal/v15/n7/full/nrc3950.html" target="_blank">2</a>)  Applications of the CRISPR–Cas9 system in cancer biology</p>
−
 
+
 
−
<h4>Inspiration</h4>
+
<p> (<a href="http://www.nature.com/nrd/journal/v14/n9/full/nrd4663.html" target="_blank">3</a>) Oncolytic viruses: a new class of immunotherapy drugs</p>
−
<p>See how other teams have described and presented their projects: </p>
+
 
−
 
+
<p> (<a href="http://www.nature.com/cgt/journal/v18/n2/pdf/cgt201066a.pdf" target="_blank">4</a>)  Targeting of tumor radioiodine therapy by expression of the sodium iodide symporter under control of the survivin promoter</p>
−
<ul>
+
 
−
<li><a href="https://2014.igem.org/Team:Imperial/Project"> Imperial</a></li>
+
<p> (<a href="10.1038/nature14299" target="_blank">5</a>) In vivo genome editing using Staphylococcus aureus Cas9. </p>
−
<li><a href="https://2014.igem.org/Team:UC_Davis/Project_Overview"> UC Davis</a></li>
+
 
−
<li><a href="https://2014.igem.org/Team:SYSU-Software/Overview">SYSU Software</a></li>
+
<p> (<a href="http://onlinelibrary.wiley.com/doi/10.1111/jipb.12152/full">6</a>) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing </p>
−
</ul>
+
 
−
 
+
<p> (<a href="http://www.nature.com/srep/2013/130911/srep02623/full/srep02623.html">7</a>) DDownregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention </p>
 +
 
 +
<p> (<a href="10.1038/nmeth.2600">8</a>) RNA-guided gene activation by CRISPR-Cas9-based transcription factors. </p>
 +
 
 +
<p> (<a href="http://pubs.acs.org/doi/full/10.1021/sb400081r">9</a>) Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas</p>
 +
 
 +
<p> (<a href="http://www.jbc.org/content/273/17/10107.full">10</a>) Generation of Constitutively Active Recombinant Caspases-3 and -6 by Rearrangement of Their Subunits</p>
 +
 
 +
<p>(<a href="http://www.sciencedirect.com/science/article/pii/0092867478900995 ">11</a>) One molecule of diphtheria toxin fragment a introduced into a cell can kill the cell</p>
 +
 
 +
<p>(<a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3449504/">12</a>) SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture </p>
 +
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Revision as of 11:16, 9 September 2015



<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">

Team:BGU Israel





    Overview


    Motivation


    Although it is one of the most researched and funded fields in medicine, cancer is still a major cause of morbidity and mortality worldwide, with 14 million new cases and over 8 million deaths per year. It is the second cause of death worldwide, and it’s responsible for quarter of the death cases among developed countries. If current trends continue, cancer will soon surpass heart disease as the leading cause of death in the U.S
    The failure of current therapies to cure cancer is due to a few reasons:
    1. Most treatments cannot distinguish precisely enough between cancer and healthy cells. Low specificity means higher toxicity and high rate of adverse effects.

    2. Cancer cells have an extremely complex pathophysiology with multiple biological pathways allowing their infinite growth and resistance to treatment. Thus, intervening with only one of this pathways, as most current therapies do, is doomed to fail.

    3. Cancer is not a single disease, but a collection of diseases arising from different genetic mutations, involving abnormal cell growth.

    Our aim , therefore, is to develop the ideal cancer therapy that is both highly specific for cancer cells, efficient, and personalized for each tumor and patient genetics.

    Boomerang


    This summer we have set our goal to design and test a synthetic machine which could distinguish individual cancer cells from healthy tissue. Our design makes sure that the function of our machine will be limited exclusively to cancer cells. Our machine does so by being operated by 2 separate cancer-specific promoters, which are highly and predominantly activated in cancer cells (1)+ (link to Results figure of TERT and survivin). By using two separate promoters we ensure that our system will be exclusively activated only in cancer cells, with minimal, if any, expression in healthy cells. Simply by changing the promoters that control the system parts, our modular system makes it easy to design the system to fit the genetic profile of each individual malignancy.

    There were several ways in which we can deliver our system in the body, and we chose AAV (Adeno Associated Virus) because of its many advantages, including low pathogenicity and mild immune response. AAV is used today in advanced clinical trials for gene therapy. The efficacy of our system will be dependent on the development of effective delivery approaches. (3).

    In our specific design for the prototype/proof-of-concept studies we use promoters which are linked to tumor proliferation (human telomerase-reverse transcriptase (hTERT) promoter) and enhanced survival (human survivin promoter), both known to be highly active in multiple cancer cell types.

    The Design

    We have constructed two separate designs, both utilizing different versions of CRISPR/Cas9 system:

    • Knock-out of genes essential for cancer cell survival (e.g., to inhibit tumor proliferation and induce apoptosis)
    • Expression of exogenous proteins which could: 1) label the tumor in a way which would enable surgeons to identify its edges for its complete removal (e.g., a chromophore), 2) lead to cancer cell death (e.g., by expression of an apoptotic protein); and 3) to produce a biomarker detectable in blood and/or urine for cancer diagnosis

    Smiley face TBD

Why Boomerang?

Like a boomerang (boomerang logo) thrown by a person which flies back instantly, our synthetic machine uses cancer cells' own genetic alterations against them.


References

(1) The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters

(2) Applications of the CRISPR–Cas9 system in cancer biology

(3) Oncolytic viruses: a new class of immunotherapy drugs

(4) Targeting of tumor radioiodine therapy by expression of the sodium iodide symporter under control of the survivin promoter

(5) In vivo genome editing using Staphylococcus aureus Cas9.

(6) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing

(7) DDownregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention

(8) RNA-guided gene activation by CRISPR-Cas9-based transcription factors.

(9) Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas

(10) Generation of Constitutively Active Recombinant Caspases-3 and -6 by Rearrangement of Their Subunits

(11) One molecule of diphtheria toxin fragment a introduced into a cell can kill the cell

(12) SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture







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