Team:Slovenia/SafetyMechanismsTermination

From 2012.igem.org

(Difference between revisions)
 
(22 intermediate revisions not shown)
Line 2: Line 2:
<head>
<head>
<meta http-equiv="X-UA-Compatible" content="IE=edge" />
<meta http-equiv="X-UA-Compatible" content="IE=edge" />
 +
 +
<!-- back to top -->
 +
<div style="position:fixed; bottom:45px; right:30px; width:100px; height:66px; background-color:transparent;">
 +
<a href="#topofthepage">
 +
<table style="background-color:transparent;" onclick="window.location = '#topofthepage'" class="invisible" style="height:100%;">
 +
<tr class="invisible" style="background-color:transparent;">
 +
<td class="invisible" style="background-color:transparent;" valign="center">
 +
<img width="100px" src ="https://static.igem.org/mediawiki/2012/1/14/Svn12_hp_btt.png"></img>
 +
</td></tr></table>
 +
</a>
 +
</div>
<style type="text/css">
<style type="text/css">
Line 201: Line 212:
#cssmenu ul li > ul li{display:block; list-style:inside none; padding:0; margin:0; position:relative;}  
#cssmenu ul li > ul li{display:block; list-style:inside none; padding:0; margin:0; position:relative;}  
#cssmenu ul li > ul li a{ outline:none; display:block; position:relative; margin:0; padding:8px 20px; font:10pt Arial, Helvetica, sans-serif; color:#fff; text-decoration:none; text-shadow:1px 1px 0 rgba(0,0,0, 0.5); }  
#cssmenu ul li > ul li a{ outline:none; display:block; position:relative; margin:0; padding:8px 20px; font:10pt Arial, Helvetica, sans-serif; color:#fff; text-decoration:none; text-shadow:1px 1px 0 rgba(0,0,0, 0.5); }  
 +
#cssmenu ul li > ul li a table tr td span{ outline:none; display:block; position:relative; margin:0; padding:0px 0px; font:10pt Arial, Helvetica, sans-serif; color:#fff; text-decoration:none; text-shadow:1px 1px 0 rgba(0,0,0, 0.5); }
#cssmenu, #cssmenu > ul > li > ul > li a:hover
#cssmenu, #cssmenu > ul > li > ul > li a:hover
{ background:#043A6B;  
{ background:#043A6B;  
Line 216: Line 228:
#cssmenu > ul > li > a:hover{background:#0C5DA5;}  
#cssmenu > ul > li > a:hover{background:#0C5DA5;}  
/* end CSS navigation menu (blue) */
/* end CSS navigation menu (blue) */
 +
 +
/*new table start*/
 +
table.newtable {background-color:transparent;}
 +
td.newtable, th.newtable {background-color:transparent;}
 +
thead.newtable{ }
 +
tbody .newtable{}
 +
/*new table start*/
 +
Line 292: Line 312:
<body>
<body>
 +
<a name="topofthepage" style="background-color:transparent;"></a>
<div id="banner">
<div id="banner">
-
<a style="position:absolute; top:0px; left:490px;" href="https://2012.igem.org/Main_Page"><b>iGEM 2012</b></a>
 
</div>
</div>
Line 316: Line 336:
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li>  
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li>  
-
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li>  
+
<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch';" class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>
 +
    <li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchControls';" class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>  
  </ul>
  </ul>
</li>
</li>
Line 324: Line 345:
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li>  
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li>  
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li>  
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li>  
-
<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><span>Microcapsule degradation</span></a></li>  
+
    <li><a href="https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation"><table  onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>  
  </ul>
  </ul>
</li>
</li>
Line 332: Line 353:
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li>  
<li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li>  
 +
    <li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>
 
 
  </ul>
  </ul>
Line 339: Line 361:
  <ul>
  <ul>
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li>
-
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><span>Pharmacokinetics</span></a></li>
+
    <li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li>
-
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><span>Quantitative and stability model</span></a></li>  
+
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>  
-
<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><span>Interactive simulations</span></a></li>
+
    <li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>
  </ul>
  </ul>
</li>
</li>
Line 355: Line 377:
  <ul>
  <ul>
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li>
-
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><span>Lablog</span></a></li>
+
    <li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li>  
<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li>  
  </ul>
  </ul>
Line 378: Line 400:
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li>
 +
<li><a href='https://2012.igem.org/Team:Slovenia/TeamCollaborations'><table  onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li>
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li>  
<li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li>  
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li>  
<li><a href='https://2012.igem.org/Team:Slovenia/TeamSponsors'><span>Sponsors</span></a></li>  
Line 385: Line 408:
</div>
</div>
<!-- end main menu -->
<!-- end main menu -->
 +
</div> <!-- end menu -->
</div> <!-- end menu -->
Line 390: Line 414:
<div id="main">
<div id="main">
<br/>
<br/>
 +
 +
 +
<!-- dummy link na bannerju -->
 +
<a href="https://2012.igem.org/Main_Page">
 +
<div id="dummy" style="background-color:transparent; position:absolute; left:870px; top:25px; width:115px; height:80px; z-index:100; opacity:0.0;">
 +
</div>
 +
</a>
<!-- VSEBINA OD TU DALJE -->
<!-- VSEBINA OD TU DALJE -->
<h1>Termination</h1>
<h1>Termination</h1>
-
<p>In order to terminate our system at the end of the therapy, we designed our therapeutic cells so that they would produce thymidine kinase.</p>
+
<table class="summary">
-
<p>Expression of thymidine kinase is not deleterious to cell growth.</p>
+
<tr class="summary">
 +
<td class="summary">
 +
<p>In order to terminate our system at the end of the therapy, we designed our therapeutic cells to produce the enzyme thymidine kinase.</p>
<p>The addition of the prodrug ganciclovir, which is converted into a toxic compound in cells producing thymidine kinase, efficiently initiates the apoptosis of therapeutic cells, making this mechanism virtualy free of leaky apoptosis.</p>
<p>The addition of the prodrug ganciclovir, which is converted into a toxic compound in cells producing thymidine kinase, efficiently initiates the apoptosis of therapeutic cells, making this mechanism virtualy free of leaky apoptosis.</p>
 +
<p>Expression of thymidine kinase is not deleterious to cell growth.</p>
 +
 +
</td>
 +
</tr>
 +
</table>
 +
<h3>The Thymidine kinase/Ganciclovir system in detail</h3>
<h3>The Thymidine kinase/Ganciclovir system in detail</h3>
<p>
<p>
-
<p>Suicide gene therapy or gene-directed enzyme prodrug therapy (GDEPT) is widely used in cancer treatment. One of the most used GDEPT systems is the <b>herpes simplex virus thymidine kinase (HSV-TK)</b> with <b>purine nucleoside analog ganciclovir (GCV)</b> as a prodrug. Systemic administration of the prodrug ganciclovir induces apoptosis only in cells transfected with HSV-thymidine kinase while the untransfected cells survive. Unlike human thymidine kinase, HSV-thymidine kinase is able to phosphorylate ganciclovir to form ganciclovir-monophosphate, which is then phosphorylated to ganciclovir-diphosphate followed by ganciclovir-triphosphate. Ganciclovir-triphosphate is then incorporated into the DNA, which causes inhibition of DNA synthesis and subsequently leads to apoptosis (Ardiani et al., 2010).  We used the HSV-thymidine kinase fused to mouse guanylate kinase (mGMK:TK30, BioBrick BBa_K404113, prepared by the Freiburg_Bioware team in 2010) in our system because it improves phosphorylation of ganciclovir-monophosphate to ganciclovir-diphosphate, thus preventing accumulation of ineffective intermediate products (i.e. GCV-MP, GCV-DP) due to the limited ability of the endogenous guanylate kinase (Figure 1).</p>
+
<p>Suicide gene therapy or gene-directed enzyme prodrug therapy (GDEPT) is widely used in cancer treatment. One of the most used GDEPT systems is the <b>herpes simplex virus thymidine kinase (HSV-TK)</b> with <b>purine nucleoside analog ganciclovir (GCV)</b> as a prodrug. Systemic administration of the prodrug ganciclovir induces apoptosis only in cells transfected with HSV-thymidine kinase while the untransfected cells survive. Unlike human thymidine kinase, HSV-thymidine kinase is able to phosphorylate ganciclovir to form ganciclovir-monophosphate, which is then phosphorylated to ganciclovir-diphosphate followed by ganciclovir-triphosphate. Ganciclovir-triphosphate is then incorporated into the DNA, which causes inhibition of DNA synthesis and subsequently leads to apoptosis (Ardiani et al., 2010).  We used the HSV-thymidine kinase fused to mouse guanylate kinase (mGMK:TK30, BioBrick <a href="http://partsregistry.org/Part:BBa_K404113">BBa_K404113</a>, prepared by the Freiburg_Bioware team in 2010) in our system because it improves phosphorylation of ganciclovir-monophosphate to ganciclovir-diphosphate, thus preventing accumulation of ineffective intermediate products (i.e. GCV-MP, GCV-DP) due to the limited ability of the endogenous guanylate kinase (Figure 1).</p>
<p>We successfully implemented HSV-TK/GCV system to our cellular device to function as a controllable “safety switch”. This means that <b>we can inactivate our cellular device whenever we want</b>, after or even in the middle of therapy, simply by administering ganciclovir to the patient.</p>
<p>We successfully implemented HSV-TK/GCV system to our cellular device to function as a controllable “safety switch”. This means that <b>we can inactivate our cellular device whenever we want</b>, after or even in the middle of therapy, simply by administering ganciclovir to the patient.</p>
-
<img src="https://static.igem.org/mediawiki/2012/8/83/Svn12_safety_mechanisms_termination_fig1.png"></img>
+
 
-
<p><b>Figure 1. Mechanism of action of the HSV-TK/GCV system.</b></p>
+
<!-- figure 1 -->
-
<p>Following the BioBrick Standard Assembly technique we inserted the mouse guanylate kinase – thymidine kinase fusion gene (mGMK:TK30, (BBa_K404113)) under the control of the CMV promoter (Figure 2).  HSV-TK mutant (TK30) contains six amino acid substitutions and shows enhanced sensitivity to ganciclovir (Kokoris et al., 1999).</p>
+
<table class="invisible" style="width:90%;">
-
<img src="https://static.igem.org/mediawiki/2012/0/05/Svn12_safety_mechanisms_termination_fig2.png"></img>
+
<tbody  class="invisible">
-
<p><b>Figure 2. Assembly of mGMK:TK30 (BBa_K404113) under the control of CMV promoter.</b> Insert (mGMK:TK30, (BBa_K404113)) was digested with EcoRI and SpeI and pcDNA3 vector with EcoRI and XbaI, loaded on an agarose gel and the appropriate bands were purified. Vector and insert were ligated and transformed into <i>E. coli</i> strain DH5α. The DNA sequence of the resulting plasmid pPCMV_mGMK:TK30 was confirmed by DNA sequencing.</p>
+
<tr class="invisible">
-
<p>We transfected HEK293 cells with the pPCMV_mGMK:TK30 plasmid and monitored cell survival after the addition of ganciclovir. We daily observed cells with an light microscope and monitored the morphological signs of decreased viability. Additionally we stained cells with Hoechst and SytoxGreen514 dye to distinguish between live and dead cells with the help of a confocal microscope. We quantified viable cell numbers at different time points by cell counting and flow cytometry. For detailed protocols please refer to the "Experimental methods" section.</p>
+
<td class="invisible">
 +
<img class="invisible" src="https://static.igem.org/mediawiki/2012/8/83/Svn12_safety_mechanisms_termination_fig1.png"/>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<table class="invisible" style="width:80%; text-align:center;">
 +
<tbody  class="invisible">
 +
<tr class="normal"><td class="invisible">
 +
<b>Figure 1. Mechanism of action of the HSV-TK/GCV system.</b>
 +
</td></tr>
 +
</tbody>
 +
</table>
 +
<!-- end table-->
 +
 
 +
 
 +
 
 +
<p>Following the BioBrick Standard Assembly technique we inserted the mouse guanylate kinase – thymidine kinase fusion gene (mGMK:TK30, (<a href="http://partsregistry.org/Part:BBa_K404113">BBa_K404113</a>)) under the control of the CMV promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782063">BBa_K782063</a>; Figure 2).  HSV-TK mutant (TK30) contains six amino acid substitutions and shows enhanced sensitivity to ganciclovir (Kokoris et al., 1999).</p>
 +
 
 +
 
 +
<!-- figure 1 -->
 +
<table class="invisible" style="width:90%;">
 +
<tbody  class="invisible">
 +
<tr class="invisible">
 +
<td class="invisible">
 +
<img class="invisible" src="https://static.igem.org/mediawiki/2012/0/05/Svn12_safety_mechanisms_termination_fig2.png"/>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<table class="invisible" style="width:70%; text-align:justify;">
 +
<tbody  class="invisible">
 +
<tr class="normal"><td class="invisible">
 +
<b>Figure 2. Assembly of mGMK:TK30 (<a href="http://partsregistry.org/Part:BBa_K404113">BBa_K404113</a>) under the control of CMV promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782063">BBa_K782063</a>).</b> Insert (mGMK:TK30, (<a href="http://partsregistry.org/Part:BBa_K404113">BBa_K404113</a>)) was digested with EcoRI and SpeI and pcDNA3 vector with EcoRI and XbaI, loaded on an agarose gel and the appropriate bands were purified. Vector and insert were ligated and transformed into <i>E. coli</i> strain DH5α. The DNA sequence of the resulting plasmid pPCMV_mGMK:TK30 was confirmed by DNA sequencing.
 +
</td></tr>
 +
</tbody>
 +
</table>
 +
<!-- end table-->
 +
 
 +
 
 +
 
 +
 
 +
<p>We transfected HEK293 cells with the pPCMV_mGMK:TK30 plasmid and monitored cell survival after the addition of ganciclovir. We daily observed cells a with light microscope and monitored the morphological signs of decreased viability. Additionally we stained cells with Hoechst and SytoxGreen514 dye to distinguish between live and dead cells with the help of a confocal microscope. We quantified viable cell numbers at different time points by cell counting and flow cytometry. For detailed protocols please refer to the <a href="https://2012.igem.org/Team:Slovenia/Notebook">Experimental methods</a> section.</p>
</p>
</p>
<h2>Results</h2>
<h2>Results</h2>
<p>
<p>
-
<p>We tested different amounts of transfected pPCMV_mGMK:TK30 DNA, different concentrations of ganciclovir and different time periods after the addition of ganciclovir to find the optimal conditions where our pCMV_mGMK:TK30-transfected cells would be most efficiently killed while the untransfected cells would be left unharmed. We monitored pPCMV_mGMK:TK30-transfected ganciclovir-treated cells under a light microscope and discovered that as little as 40 hours of incubation with higher concentrations of ganciclovir (>30 μg/ml) is enough to kill over 50 % of our pPCMV-mGMK:TK30-transfected cells. After 65 hours of incubation with ganciclovir there was a decrease in cell number and survival already at low concentration of ganciclovir (1 μg/ml). We showed that the HSV-TK/GCV system successfully kills cells even at low amounts of transfected mGMK:TK30 and upon addition of relatively low concentrations of ganciclovir (5 μg/ml).</p>
+
<p>We tested different amounts of transfected pPCMV_mGMK:TK30 DNA, different concentrations of ganciclovir and different time periods after the addition of ganciclovir to find the optimal conditions where our pCMV_mGMK:TK30-transfected cells would be most efficiently killed while the untransfected cells would be left unharmed. We monitored pPCMV_mGMK:TK30-transfected ganciclovir-treated cells under a light microscope and discovered that as little as 40 hours of incubation with higher concentrations of ganciclovir (>30 µg/ml) is enough to kill over 50 % of our pPCMV-mGMK:TK30-transfected cells. After 65 hours of incubation with ganciclovir there was a decrease in cell number and survival already at low concentrations of ganciclovir (1 µg/ml). We showed that the HSV-TK/GCV system successfully kills cells even at low amounts of transfected mGMK:TK30 and upon addition of relatively low concentrations of ganciclovir (5 µg/ml).</p>
-
<p>We determined the number of viable cells after ganciclovir treatment by staining the cells with Trypan Blue (to distinguish live cells from the dead) and counting them. After 6 days of incubation with ganciclovir, the cytotoxic effect could be seen when only 1 μg/ml ganciclovir was added to pPCMV_mGMK:TK30 transfected cells while untransfected cells remained unharmed. The number of viable cells decreased to <10 % when 10 μg/ml ganciclovir was added. These results demonstrate that our safety mechanism is effective even at low doses of ganciclovir, when only therapeutic cells are killed while untransfected cells are left unharmed.</p>
+
<p>We determined the number of viable cells after ganciclovir treatment by staining the cells with Trypan Blue (to distinguish between living and dead cells) and counting them. After 6 days of incubation with ganciclovir, the cytotoxic effect could be seen when only 1 µg/ml ganciclovir was added to pPCMV_mGMK:TK30 transfected cells while untransfected cells remained unharmed. The number of viable cells decreased to <10 % when 10 µg/ml ganciclovir was added. These results demonstrate that our safety mechanism is effective even at low doses of ganciclovir, where only therapeutic cells are killed while other cells are left unharmed.</p>
 +
 
 +
 
 +
 
 +
<!-- figure 1 -->
 +
<table class="invisible" style="width:90%;">
 +
<tbody  class="invisible">
 +
<tr class="invisible">
 +
<td class="invisible">
 +
<img  class="invisible" src="https://static.igem.org/mediawiki/2012/c/cd/Svn12_safety_mechanisms_termination_fig3.png"/>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<table class="invisible" style="width:80%; text-align:justify;">
 +
<tbody  class="invisible">
 +
<tr class="normal"><td class="invisible">
 +
<b>Figure 3. Cytotoxic effect of ganciclovir (GCV) on with mGMK:TK30 transfected cells.</b> HEK293 cells were transfected with different amounts of plasmid PCMV_mGMK:TK30 and treated with ganciclovir (1, 10 and 30 mg/L). Cell survival was analysed after 6 days of incubation with GCV.
 +
</td></tr>
 +
</tbody>
 +
</table>
 +
<!-- end table-->
 +
 
 +
 
 +
<!-- figure -->
 +
<table class="invisible" style="width:90%;">
 +
<tbody  class="invisible">
 +
<tr class="invisible">
 +
<td class="invisible">
 +
<img  class="invisible" src="https://static.igem.org/mediawiki/2012/c/c3/Svn12_safety_mechanisms_termination_fig4.png"/>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<table class="invisible" style="width:80%; text-align:justify;">
 +
<tbody  class="invisible">
 +
<tr class="normal"><td class="invisible">
 +
<b>Figure 4. HEK293 cells with or without mGMK:TK30 after 5 days of incubation with ganciclovir (GCV).</b> Cells were stained with Hoechst (blue) and SytoxGreen514 dye (dead/pink). (A-C) Untransfected cells: without GCV (A) or with 10 µg/ml (B) or 100 µg/ml (C) GCV show a high survival rate. (D) Cells transfected with 200ng pPCMV_mGMK:TK30 and without GCV show high cell viability. (E-F) Cells transfected with 200 ng pPCMV_mGMK:TK30 and incubated with 10 µg/ml (E) or 100 µg/ml (F) GCV exhibit dramatically lower viability.
 +
</td></tr>
 +
</tbody>
 +
</table>
 +
<!-- end table-->
 +
<p>Cells were stained with Hoechst and SytoxGreen514 dye that differentially stain live and dead cells (Figure 4) and were imaged with a confocal microscope. Cell permeable Hoechst (depicted blue) binds to nucleic acids and therefore stains all cells in a culture. On the other hand cell impermeable SytoxGreen (depicted pink) cannot cross the membrane of live cells and therefore stains only dead cells. The cytotoxic effect of ganciclovir is evident at concentrations as low as 10 µg/ml and only a few viable cells were present upon addition of 100 µg/ml ganciclovir. On the other hand untransfected cells are not affected by ganciclovir. Moreover, cells expressing mGMK:TK30 (under the control of the CMV promoter) had no effect on cell survival if no ganciclovir was added.</p>
 +
 
 +
 
 +
 
 +
<p>The bystander effect for mGMK:TK30 was demonstrated by Ardiani et al. (2010) who showed substantial ganciclovir-induced cell death although only a fraction of cells were transfected. There are several different mechanisms of a bystander effect: the transfer of toxic metabolite ganciclovir-triphosphate through gap junctions (Elshami et al., 1996), endocytosis of apoptotic bodies generated from HSV-TK expressing cells by neighbouring untransfected cells (Freeman et al., 1993) and release of soluble factors (Drake et al., 2000). We also demonstrated that even if some cells do not express mGMK:TK30 (GFP was used as a control of transfection), ganciclovir affects practically all cells in a culture (Figure 5). If transiently transfected therapeutic cells would be microencapsulated and used for therapy, the bystander effect ensures that all cells in a microcapsule would be killed upon addition of ganciclovir even if some of them would not express the HSV-thymidine kinase.</p>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<!-- figure -->
 +
<table class="invisible" style="width:90%;">
 +
<tbody  class="invisible">
 +
<tr class="invisible">
 +
<td class="invisible">
 +
<img  class="invisible" src="https://static.igem.org/mediawiki/2012/3/39/Svn12_safety_mechanisms_termination_fig5.png"/>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<table class="invisible" style="width:80%; text-align:justify;">
 +
<tbody  class="invisible">
 +
<tr class="normal"><td class="invisible">
 +
<b>Figure 5. Ganciclovir kills cells expressing mGMK:TK30.</b> Cells were incubated with GCV for 5 days. Cells were transfected with 100 ng plasmid PCMV_mGMK:TK30 and 20 ng GFP (depicted green). Cells depicted on (A) were not treated with GCV. Image (B) depicts cell death after incubation with 10 µg/mL GCV and image (C) shows drastic decrease in cell survival after incubation with 100 µg/mL GCV.
 +
</td></tr>
 +
</tbody>
 +
</table>
 +
<!-- end table-->
 +
 
 +
<p>Trypan Blue and SytoxGreen514 only stain cells that are already dead. We also wanted to determine the number of cells that are still in the early stages of apoptosis. To do this we stained the cells with Annexin V and analyzed them with flow cytometry. Annexin V binds phosphatidylserine, which is located on the inner side of the plasma membrane. When apoptosis is initiated phosphatidylserine is translocated to the extracellular side of the plasma membrane which enables staining with Annexin V.</p>
 +
<p>Figure 6 shows the percent of Annexin V positive cells 65 hours after ganciclovir addition. The percent of apoptotic cells increases with higher ganciclovir concentrations and higher amounts of transfected pPCMV_mGMK:TK30. Even though only around 25 % of cells stain with Annexin V we must keep in mind that after 65 hours ganciclovir already killed a large amount of cells which were therefore not included in the analysis.</p>
 +
 
 +
 
 +
 
 +
 
 +
<!-- figure -->
 +
<table class="invisible" style="width:90%;">
 +
<tbody  class="invisible">
 +
<tr class="invisible">
 +
<td class="invisible">
 +
<img  class="invisible" src="https://static.igem.org/mediawiki/2012/0/01/Svn12_safety_mechanisms_termination_fig6.png"/>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<table class="invisible" style="width:80%; text-align:justify;">
 +
<tbody  class="invisible">
 +
<tr class="normal"><td class="invisible">
 +
<b>Figure 6. Apoptosis triggered by ganciclovir is mGMK:TK30 dependent.</b> Analysis by flow cytometry was conducted on untransfected cells and cells transfected with 500 ng and 1000 ng plasmid PCMV_mGMK:TK30, after 65 hours of incubation with 0, 30 and 100 mg/L GCV. Cells were gated according to the intensity of Annexin V-PE (channel FL2) compared to unstained cells.
 +
</td></tr>
 +
</tbody>
 +
</table>
 +
<!-- end table-->
 +
 
<h2 style="color:grey;">References</h2>
<h2 style="color:grey;">References</h2>
<p style="color:grey;">
<p style="color:grey;">
-
Bauer, S., Groh, V., Wu, J., Steinle, A., Phillips, J.H., Lanier, L.L., Spies, T. (1999) Activation of NK Cells and T Cells by NKG2D, a receptor for Stress-Inducible MICA. <i>Science</i> <b>285</b>, 727-729.<br/><br/>
+
Ardiani, A., Sanchez-bonilla, M., and Black, M.E. (2010) Fusion Enzymes Containing HSV-1 Thymidine Kinase Mutants and Guanylate Kinase Enhance Prodrug Sensitivity In Vitro and In Vivo. <i>Cancer</i> <b>17</b>, 86-96.<br/><br/>
-
Borrego, F., Kabat, J., Kim, D.K., Lieto, L., Maasho, K., Peña, J., Solana, R., Coligan J.E. (2001) Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells. <i>Mol. Immunol.</i> <b>38</b>, 637-660.<br/><br/>
+
Drake, R.R., Pitlyk, K., McMasters, R.A., Mercer, K.E., Young, H., Moyer, M.P. (2000) Connexin-independent ganciclovir-mediated killing conferred on bystander effect-resistant cell lines by a herpes simplex virus-thymidine kinase-expressing colon cell line. <i>Molecular therapy: the journal of the American Society of Gene Ther.</i> <b>2</b>, 515-523.<br/><br/>
-
Groh, V., Rhinehart, R., Secrist, H., Bauer., S., Grabstein, K.H., Spies, T. (1999) Broad tumor-associated expression and recognition by tumor-derived gd T cells of MICA and MICB. <i>Proc. Natl. Acad. Sci.</i> <b>96</b>, 6879–6884.<br/><br/>
+
Elshami, A.A., Saavedra, A., Zhang, H., Kucharczuk, J.C., Spray, D.C., Fishman, G.I., Amin, K.M., Kaiser, L.R., Albelda, S.M. (1996) Gap junctions play a role in the ‘bystander effect’ of the herpes simplex virus thymidine kinase/ganciclovir system in vitro. <i>Gene Ther.</i> <b>3</b>, 85-92.<br/><br/>
-
Salih, H.R., Rammensee, H.G., Steinle, A. (2002) Cutting Edge: Down-Regulation of MICA on Human Tumors by Proteolytic Shedding. <i>J Immunol.</i> <b>169</b>, 4098-4102.<br/><br/>
+
Freeman, S.M., Abboud, C.N., Whartenby, K.A., Packman, C.H., Koeplin, D.S., Moolten, F.L., Abraham, G.N.  (1993) The Bystander Effect : Tumor Regression When a Fraction of the Tumor Mass Is Genetically Modified. <i>In Vitro</i> <b>53</b>, 5274-5283.<br/><br/>
-
Stenile, A., Li, P., Morris, D.L., Groh, V., Lanier, L.L., Strong, R.K., Spies, T. (2001) Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family. <i>Immunogenet.</i> <b>53</b>, 279-287.
+
Kokoris, M.S., Sabo P., Adman E.T., Black, M.E. (1999) Enhancement of tumor ablation by a selected HSV-1 thymidine kinase mutant. <i>Gene Ther.</i> <b>6</b>, 1415-1426.
</p>
</p>
 +
 +
 +
 +
<hr>
 +
<b>
 +
Next: <a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'>Microcapsule degradation >></a>
 +
</b>
<!-- KONEC VSEBINE -->
<!-- KONEC VSEBINE -->

Latest revision as of 21:07, 26 October 2012


Termination

In order to terminate our system at the end of the therapy, we designed our therapeutic cells to produce the enzyme thymidine kinase.

The addition of the prodrug ganciclovir, which is converted into a toxic compound in cells producing thymidine kinase, efficiently initiates the apoptosis of therapeutic cells, making this mechanism virtualy free of leaky apoptosis.

Expression of thymidine kinase is not deleterious to cell growth.

The Thymidine kinase/Ganciclovir system in detail

Suicide gene therapy or gene-directed enzyme prodrug therapy (GDEPT) is widely used in cancer treatment. One of the most used GDEPT systems is the herpes simplex virus thymidine kinase (HSV-TK) with purine nucleoside analog ganciclovir (GCV) as a prodrug. Systemic administration of the prodrug ganciclovir induces apoptosis only in cells transfected with HSV-thymidine kinase while the untransfected cells survive. Unlike human thymidine kinase, HSV-thymidine kinase is able to phosphorylate ganciclovir to form ganciclovir-monophosphate, which is then phosphorylated to ganciclovir-diphosphate followed by ganciclovir-triphosphate. Ganciclovir-triphosphate is then incorporated into the DNA, which causes inhibition of DNA synthesis and subsequently leads to apoptosis (Ardiani et al., 2010). We used the HSV-thymidine kinase fused to mouse guanylate kinase (mGMK:TK30, BioBrick BBa_K404113, prepared by the Freiburg_Bioware team in 2010) in our system because it improves phosphorylation of ganciclovir-monophosphate to ganciclovir-diphosphate, thus preventing accumulation of ineffective intermediate products (i.e. GCV-MP, GCV-DP) due to the limited ability of the endogenous guanylate kinase (Figure 1).

We successfully implemented HSV-TK/GCV system to our cellular device to function as a controllable “safety switch”. This means that we can inactivate our cellular device whenever we want, after or even in the middle of therapy, simply by administering ganciclovir to the patient.

Following the BioBrick Standard Assembly technique we inserted the mouse guanylate kinase – thymidine kinase fusion gene (mGMK:TK30, (BBa_K404113)) under the control of the CMV promoter (BBa_K782063; Figure 2). HSV-TK mutant (TK30) contains six amino acid substitutions and shows enhanced sensitivity to ganciclovir (Kokoris et al., 1999).

We transfected HEK293 cells with the pPCMV_mGMK:TK30 plasmid and monitored cell survival after the addition of ganciclovir. We daily observed cells a with light microscope and monitored the morphological signs of decreased viability. Additionally we stained cells with Hoechst and SytoxGreen514 dye to distinguish between live and dead cells with the help of a confocal microscope. We quantified viable cell numbers at different time points by cell counting and flow cytometry. For detailed protocols please refer to the Experimental methods section.

Results

We tested different amounts of transfected pPCMV_mGMK:TK30 DNA, different concentrations of ganciclovir and different time periods after the addition of ganciclovir to find the optimal conditions where our pCMV_mGMK:TK30-transfected cells would be most efficiently killed while the untransfected cells would be left unharmed. We monitored pPCMV_mGMK:TK30-transfected ganciclovir-treated cells under a light microscope and discovered that as little as 40 hours of incubation with higher concentrations of ganciclovir (>30 µg/ml) is enough to kill over 50 % of our pPCMV-mGMK:TK30-transfected cells. After 65 hours of incubation with ganciclovir there was a decrease in cell number and survival already at low concentrations of ganciclovir (1 µg/ml). We showed that the HSV-TK/GCV system successfully kills cells even at low amounts of transfected mGMK:TK30 and upon addition of relatively low concentrations of ganciclovir (5 µg/ml).

We determined the number of viable cells after ganciclovir treatment by staining the cells with Trypan Blue (to distinguish between living and dead cells) and counting them. After 6 days of incubation with ganciclovir, the cytotoxic effect could be seen when only 1 µg/ml ganciclovir was added to pPCMV_mGMK:TK30 transfected cells while untransfected cells remained unharmed. The number of viable cells decreased to <10 % when 10 µg/ml ganciclovir was added. These results demonstrate that our safety mechanism is effective even at low doses of ganciclovir, where only therapeutic cells are killed while other cells are left unharmed.

Cells were stained with Hoechst and SytoxGreen514 dye that differentially stain live and dead cells (Figure 4) and were imaged with a confocal microscope. Cell permeable Hoechst (depicted blue) binds to nucleic acids and therefore stains all cells in a culture. On the other hand cell impermeable SytoxGreen (depicted pink) cannot cross the membrane of live cells and therefore stains only dead cells. The cytotoxic effect of ganciclovir is evident at concentrations as low as 10 µg/ml and only a few viable cells were present upon addition of 100 µg/ml ganciclovir. On the other hand untransfected cells are not affected by ganciclovir. Moreover, cells expressing mGMK:TK30 (under the control of the CMV promoter) had no effect on cell survival if no ganciclovir was added.

The bystander effect for mGMK:TK30 was demonstrated by Ardiani et al. (2010) who showed substantial ganciclovir-induced cell death although only a fraction of cells were transfected. There are several different mechanisms of a bystander effect: the transfer of toxic metabolite ganciclovir-triphosphate through gap junctions (Elshami et al., 1996), endocytosis of apoptotic bodies generated from HSV-TK expressing cells by neighbouring untransfected cells (Freeman et al., 1993) and release of soluble factors (Drake et al., 2000). We also demonstrated that even if some cells do not express mGMK:TK30 (GFP was used as a control of transfection), ganciclovir affects practically all cells in a culture (Figure 5). If transiently transfected therapeutic cells would be microencapsulated and used for therapy, the bystander effect ensures that all cells in a microcapsule would be killed upon addition of ganciclovir even if some of them would not express the HSV-thymidine kinase.

Trypan Blue and SytoxGreen514 only stain cells that are already dead. We also wanted to determine the number of cells that are still in the early stages of apoptosis. To do this we stained the cells with Annexin V and analyzed them with flow cytometry. Annexin V binds phosphatidylserine, which is located on the inner side of the plasma membrane. When apoptosis is initiated phosphatidylserine is translocated to the extracellular side of the plasma membrane which enables staining with Annexin V.

Figure 6 shows the percent of Annexin V positive cells 65 hours after ganciclovir addition. The percent of apoptotic cells increases with higher ganciclovir concentrations and higher amounts of transfected pPCMV_mGMK:TK30. Even though only around 25 % of cells stain with Annexin V we must keep in mind that after 65 hours ganciclovir already killed a large amount of cells which were therefore not included in the analysis.

References

Ardiani, A., Sanchez-bonilla, M., and Black, M.E. (2010) Fusion Enzymes Containing HSV-1 Thymidine Kinase Mutants and Guanylate Kinase Enhance Prodrug Sensitivity In Vitro and In Vivo. Cancer 17, 86-96.

Drake, R.R., Pitlyk, K., McMasters, R.A., Mercer, K.E., Young, H., Moyer, M.P. (2000) Connexin-independent ganciclovir-mediated killing conferred on bystander effect-resistant cell lines by a herpes simplex virus-thymidine kinase-expressing colon cell line. Molecular therapy: the journal of the American Society of Gene Ther. 2, 515-523.

Elshami, A.A., Saavedra, A., Zhang, H., Kucharczuk, J.C., Spray, D.C., Fishman, G.I., Amin, K.M., Kaiser, L.R., Albelda, S.M. (1996) Gap junctions play a role in the ‘bystander effect’ of the herpes simplex virus thymidine kinase/ganciclovir system in vitro. Gene Ther. 3, 85-92.

Freeman, S.M., Abboud, C.N., Whartenby, K.A., Packman, C.H., Koeplin, D.S., Moolten, F.L., Abraham, G.N. (1993) The Bystander Effect : Tumor Regression When a Fraction of the Tumor Mass Is Genetically Modified. In Vitro 53, 5274-5283.

Kokoris, M.S., Sabo P., Adman E.T., Black, M.E. (1999) Enhancement of tumor ablation by a selected HSV-1 thymidine kinase mutant. Gene Ther. 6, 1415-1426.


Next: Microcapsule degradation >>