Team:Slovenia/ImplementationIschaemicHeartDisease

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<a style="position:absolute; top:0px; left:490px;" href="https://2012.igem.org/Main_Page"><b>iGEM 2012</b></a>
 
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<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>  
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<li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li>  
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<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>
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    <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>
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<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>  
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<li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation'><span>Microcapsule degradation</span></a></li>  
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    <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>
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<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>  
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    <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>
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  <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>
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<li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><span>Pharmacokinetics</span></a></li>
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    <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>
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<li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><span>Quantitative and stability model</span></a></li>  
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<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>  
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<li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><span>Interactive simulations</span></a></li>
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    <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>
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  <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>
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<li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><span>Lablog</span></a></li>
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    <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>
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<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>
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<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>  
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<h1>Hepatitis C</h1>
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<h1>Ischaemic heart disease</h1>
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<p>We designed a device for the therapy of hepatitis C, composed of microencapsulated mammalian cells that include a genetic bistable toggle switch with a positive feedback loop, where in one state the cells produce interferon alpha (IFN-α) as the antiviral effector and in the second state they produce hepatocyte growth factor (HGF) to promote liver regeneration.</p>
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<p>A pharmacokinetic model demonstrated that if the device is implanted into the liver it results in higher levels of IFN-α within the liver than systemically. More importantly, this type of application avoids the spikes of high IFN-α concentration that occur in treatment with IFN-α injections. This should decrease the severity of side effects of IFN-α experienced by a high percentage of patients.</p>
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<p>We estimated, based on the detection of IFN-α produced by HEK293 cells, that sufficient quantities of the therapeutic protein could be produced by the amount of microencapsulated cells feasible in a real therapeutic application.</p>
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<img src="https://static.igem.org/mediawiki/2012/8/8e/Svn12_implementation_hepatitis_c_fig1.png"></img>
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<p><b>Figure 1.  Therapy of hepatitis C by microencapsulated cells which can be regulated to produce and release therapeutic proteins into the liver tissue.</b></p>
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<img src="https://static.igem.org/mediawiki/2012/f/f2/Svn12_implementation_hepatitis_c_fig2.png"></img>
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<p><b>Figure 2. Scheme of the constructs for the regulated therapy of hepatitis C with interferon alpha (IFN-α) and hepatocyte growth factor (HGF).</b> Each of the therapeutic effector is released in equimolar amount to the autoactivator.</p>
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<h3>Hepatitis C virus infection</h3>
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Hepatitis C is an infectious disease caused by the <b>hepatitis C virus (HCV)</b> which primarily infects the liver.  Hepatitis C is a serious worldwide health problem with a prevalence of <b>3% in the world’s population</b>. According to WHO, 170 million individuals are infected with an incidence of 3 to 4 million new cases per year. More than 350,000 people die yearly from hepatitis C-related diseases. In the US the HCV has surpassed HIV as a cause of death. Medical care costs associated with treatment of HCV infection are estimated to be more than $600 million per year just in the USA.</p>
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<img src="https://static.igem.org/mediawiki/2012/6/64/Svn12_implementation_hepatitis_c_fig3.png"></img>
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<p><b>Figure 3. Hepatitis C prevalence</b> (<a href="http://en.wikipedia.org/wiki/File:HCV_prevalence_1999.png">http://en.wikipedia.org/wiki/File:HCV_prevalence_1999.png</a>).</p>
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<p>HCV accounts for 20% of all cases of acute hepatitis which is usually asymptomatic. In approximately 75% – 85% of patients, HCV persists as a chronic infection, placing infected persons at risk for developing <b>chronic liver disease</b>. The risk of liver <a href="http://en.wikipedia.org/wiki/Cirrhosis">cirrhosis</a> after 20 years of persistent hepatitis C infection is approximately 10-15% for men and 1-5% for women. Once cirrhosis is established, the rate of developing <a href="http://en.wikipedia.org/wiki/Hepatocellular_carcinoma">liver cancer</a> is 1 to 4% per year (Pawlotsky, 2004). It is the most common indication for orthotopic <a href="http://emedicine.medscape.com/article/431783-overview">liver transplantation</a> in the United States.</p>
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<p>Hepatitis C virus is a small, spherical, enveloped, single-stranded RNA virus, a member of the <i>Flaviviridae</i> family. Because the HCV RNA-dependent RNA polymerase lacks proofreading capabilities, it generates a large number of mutant viruses known as quasispecies when the virus replication takes place. These HCV quasispecies represent a major challenge to immune-mediated control of HCV and may explain the difficulties in vaccine development.</p>
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<h3>Current therapy</h3>
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<p>Patients with acute hepatitis C virus infection which has not resolved after 2-4 months are treated with standard <b>interferon (IFN)</b> therapy for 6 months. Treatment of chronic HCV infection has 2 goals: sustained eradication of HCV and prevention of chronic complications. <b>IFN-α</b> has been used since the 1980s in the treatment of chronic hepatitis and still represents an important part of the management of chronic hepatitis C infection. Initial studies used IFN-α monotherapy, but current treatments are a combination therapy consisting of ribavirin and IFN-alpha (Feld and Hoofnagle, 2005). <b>Side effects</b> of treatment are very common, with half of the patients suffering from <a href="http://en.wikipedia.org/wiki/Influenza-like_illness">flu like symptoms</a> and a third experiencing emotional problems. Anxiety, sleep disorders and irritability are frequently observered and can in some cases lead to severe behavioral or psychological disorders. This can in turn lead to the discontinuation of therapy.</p>
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<h3>Implementation of the microencapsulated synthetic cellular device for the therapy of hepatitis C</h3>
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<p>With our biological delivery system we aim to overcome some of the adverse effects of the IFN therapy and make the therapy more efficient and affordable. Because of the local (intrahepatic/intraperitoneal) production of IFN-α from the cell-delivery system, a lower systemic concentration is expected, which could diminish systemic side effects. Furthermore, because repetitive subcutaneous application would no longer be needed, the quality of life of patients would improve as well as compliance to the therapy.</p>
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<p>In addition to antiviral activity the design of our system allows us to include additional effector proteins in the therapy. Since the advanced stages of hepatitis seriously damage the liver, we decided to include a therapeutic drug to improve liver regeneration. <b>Hepatocyte growth factor (HGF)</b> is a paracrine cellular growth, motility and morphogenic factor secreted by mesenchymal cells. It targets and acts primarily upon epithelial and endothelial cells, but also acts on haemopoietic progenitor cells. It has been shown to have a major role in adult organ regeneration and in wound healing. HGF induces proliferation and regeneration of hepatocytes, and its application by gene therapy in several preclinical studies has <b>inhibited fibrogenesis and hepatocyte apoptosis and produced the complete resolution of fibrosis in the cirrhotic liver</b> (Ueki et al., 1999). The inclusion of HGF to the interferon therapy of hepatitis C could improve liver regeneration as HGF also promotes differentiation of stem cells into hepatocytes (Oyagi et al., 2006, Nakamure ate al., 2011).</p>
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<p>We adapted our therapeutic device for the therapy of HCV infection to include the coding regions for IFN-alpha2 and HGF coupled via a t2a peptide sequence to the activators TALA and TALB of our bistable toggle switch with a positive feedback loop. This arrangement allows the initial production of IFN-α to treat the viral infection, and an induced switch to the second state after the virus has been cleared allows improved liver regeneration with HGF production.</p>
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<h2>Pharmacokinetic modeling of the distribution of IFN-α in the therapy</h2>
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<p>Consultation with Peter Popovič, MD, Msc, an interventional radiologist, suggested that microcapsules could be applied to the liver artery via a catheter to distribute production of IFN-α and later HGH throughout the liver.</p>
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<p>A pharmacokinetic model of the localized production of IFN-α in the liver was performed based on parameters from the available literature. The model is described in details in the <a href="https://2012.igem.org/Team:Slovenia/ModelingPK">Modeling section</a>. Modeling demonstrated therapy which consists of injecting IFN-α three times a week, exhibits peaks and dips in concentration of the drug in the liver as well as in plasma. The model of our new approach demonstrates that the time course of IFN-α released by the implanted microcapsules is very stable. However the systemic level of IFN-α is not much lower than the concentration in the liver (70% of the latter) because the liver is an organ that is strongly perfused with blood. This can be observed on the animation shown bellow (Figure 4).</p>
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<p><b>Figure 4. Distribution of IFN-α in different organs of the body based on the pharmacokinetic model for the therapy of hepatitis C.</b></p>
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<h2>Production of IFN-α</h2>
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<p>We tested the biological activity of IFN-α produced by HEK293T cells from our construct to get an estimate if the production level could reach the sufficient level. Stably transfected  therapeutic cells could be selected for high production for the real therapy. Type I interferons (IFN-α and β) mediate signaling through the IFNAR receptor, the STAT1 and STAT2 components of the JAK/STAT-signal transduction pathways and finally, the interferon stimulated response element (ISRE). We used a STAT1/STAT2-responsive luciferase construct that encodes the firefly luciferase reporter gene under the control of a minimal promoter and tandem repeats of ISRE.</p>
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<p>We designed the experiment as a co-culture of HEK293T cells transfected with either the IFN-α encoding plasmid or an empty vector for control and HEK293T cells transfected with the reporter vector. Additionally, we performed a co-transfection experiment, where HEK293T cells were transfected with both the reporter and the IFN-α encoding plasmids (Figure 5). As a positive control, we used the response of the same reporter to recombinant IFN-β.</p>
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<img src="https://static.igem.org/mediawiki/2012/8/8c/Svn12_implementation_hepatitis_c_fig5.png"></img>
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<p><b>Figure 5. Expressed and secreted  IFN-α induces expression of an ISRE-dependant reporter.</b> A co-culture of HEK293T cells transfected with either the IFN-α producing plasmid or an empty vector and HEK293T cells transfected with firefly luciferase reporter with ISRE was prepared. Additionally, HEK293T cells were cotransfected with both the reporter and the IFN-α prodiucing or control plasmids. After 24 hours of incubation, a dual luciferase reporter assay was performed.</p>
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<p>To asses the required quantity of microcapsules tomachieve the therapeutic amount of IFN-α, we performed an enzyme-linked immunosorbent assay (ELISA) of the <b>IFN-α production</b> of HEK293T cells. We determined that the average production of IFN-α in a single cell in 24 hours is approximately 4,6 *10^-9 µg  . Pharmacokinetic modeling predicted the required production of interferon to achieve the theraopeutic concentration in the liver to be around <b>3 µg per day</b>. Cosidering that one capsule contains approximately 3000 cells, we would have <b>to implant 230,000 capsules into the liver</b>. This number of capsules occupies roughly <b>a volume of 15 mL</b>. We believe that this is a feasible amount for the delivery of synthetic devices into the body. Furthermore, we believe that the production of protein could be significantly increased as HEK293 cells can produce as much as 300 mg of IFN-α per liter of serum-free medium (Loignon et al., 2008).</p>
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<p>We designed a device for the therapy of myocardial ischaemia, composed of microencapsulated mammalian cells that include a genetic bistable toggle switch with a positive feedback loop, where in the first state cells produce anakinra as the anti-inflammatory effector and in the second state they produce a stoichiometric amount of the vascular endothelial growth factor (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782061">VEGF</a>) and platelet-derived growth factor B (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782061">PDGF-BB</a>) to promote angiogenesis in the damaged tissue.</p>
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<p>A pharmacokinetic model demonstrated that implantation of the device into the injured heart tissue results in a high level of anakinra concentration within the affected tissue, while the systemic level of anakinra is negligible, preventing systemic immunosuppression.</p>
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<p>We demonstrated that the production level of anakinra by engineered cells is sufficient for the therapeutic implementation of microencapsulated cells for this indication.</p>
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<tr class="invisible"><td class="invisible"><b>Figure 1. Regulated release of therapeutic proteins into the tissue.</b> 
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<b>Figure 2. Scheme of the constructs for the regulated therapy of ischaemia with IL-1Ra (IL-1 receptor antagonist, anakinra) and VEGF/PDGF-BB to suppress inflammation and promote angiogenesis, respectively.</b>
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<h3>Ischaemic heart disease</h3>
 +
<p>Ischaemic heart disease is characterized by a reduced blood supply to the heart muscle, usually due to coronary artery disease (atherosclerosis of the coronary arteries). Ischaemic heart disease (which includes myocardial infarction, angina pectoris and heart failure when preceded by myocardial infarction) is <b>the leading cause of mortality in most Western countries</b>. Menzin et al. reported a total first-year cost average of 32,345 $ and as much as 61% of these costs were due to rehospitalisation (Menzin et al., 2008). Furthermore, survivors of acute myocardial infarction remain at high risk of death in the years after the event (Grothusen et al., 2012).</p>
 +
<h4>Current therapy and research</h4>
 +
<p><b>Acute myocardial infarction</b> due to total occlusion of a coronary artery is treated with early reperfusion strategies to minimize the developing ischaemic myocardial damage. Current guidelines recommend interventional treatment via <b>percutaneous transluminal coronary angioplasty (PTCA)</b> of the occluded coronary artery followed by a stent implantation in the setting of acute myocardial infarction. In case of PTCA contraindications, <b>operative revascularization therapy CABG</b> (coronary artery bypass graft surgery) is recommended. However, many studies have shown a negative effect of reperfusion, resulting in additional myocardial injury due to activation of an inflammatory response (Grothusen et al., 2012; Abbate et al., 2008).</p>
 +
<p>Interleukin-1a and b are one of the most important inflammatory cytokines which are responsible for leukocyte chemotaxis, macrophage activation, reactive oxygen species formation, endothelial dysfunction, and cardiomyocyte apoptosis. Thus, <b>inhibition of IL-1 receptor complex activation</b> can present a new important treatment target (Grothusen et al., 2012). Furthermore, recent studies have shown the <b>cardioprotective properties of anakinra</b>, which is a nonglycosylated, human recombinant competitive inhibitor of IL-1a and b signaling through binding to the IL-1 receptor complex (Abbate et al., 2008).</p>
 +
<p>There is also experimental evidence of the important <b>regenerating role of vascular endothelial growth factor (VEGF)</b> after acute myocardial infarction (Banfi et al., 2012; Zhang et al., 2008). After binding to its receptor VEGFR1 and VEGFR2 it supports angiogenesis, inhibits endothelial cell apoptosis, promotes endothelial cell proliferation, and restores heart function. Although therapeutic angiogenesis by delivery of vascular growth factors is an attractive strategy, many clinical trials have thus far failed to show efficiency. One of the most likely explanations for this discrepancy is that VEGF induces growth of dysfunctional vessels, if expressed outside of a narrow dosage window (Banfi et al., 2012). Banfi et al. confirmed the hypothesis that <b>co-delivery of platelet-derived growth factor-BB (PDGF-BB)</b>, which recruits pericytes, induced <b>normal angiogenesis</b> in skeletal muscle irrespective of VEGF levels. It was also shown that coexpression of VEGF and PDGF-BB encoded by separate vectors in different cells or in the same cells only partially corrected aberrant angiogenesis. In marked contrast, coexpression of both factors in every cell at a fixed relative level via a single bicistronic vector led to robust, uniformly normal angiogenesis, even when VEGF expression was high and heterogeneous. Secondly, a challenge has been that with the conventional gene transfer vectors, the growth factor concentration in target tissues had not reached sufficient levels or had not persisted long enough for triggering relevant vascular growth (Zhang et al., 2008). Cell transplantation strategies emerged as a promising approach to overcome this issue.</p>
 +
<p>Zhang et al. reported the first transplantation of microencapsulated engineered xenogeneic CHO cells in post-infarction myocardium, which showed that the supplementation of VEGF from implanted xenogeneic cells could foster the formation of arterial collaterals, improve myocardial perfusion and thus also promote the regeneration of damaged myocardium augmented angiogenesis and improved heart function (Zhang et al., 2008).</p>
 +
<h4>Implementation of the microencapsulated engineered cellular device for the therapy of ischaemic heart disease</h4>
 +
<p>With our biological delivery system for biopharmaceuticals, we aim to improve the therapy of ischaemic heart disease by delivering multiple biopharmaceuticals in different time periods controlled by a physician. The encapsulated cells would be locally administered with intracardial injections via ventriculography, or intraoperatively in the proximity of the myocardial damage (Figure 1). <b>In the first phase</b>, after myocardial damage has occurred and reperfusion injury is expected, production of <b>anakinra</b> could halt the inflammatory response. <b>In the second phase</b>, the therapy would switch to the production of <b><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782061">VEGF and PDGF-BB</a></b> at stoichiometric concentrations, which could enhance the production of normal, robust vessels (Figure 2). After the therapeutic effect is reached, we could discontinue the synthesis of growth factors through the ability to control the viability of encapsulated allogenic cells. This could prevent the negative side effects of over-production of vascular growth factors (haemangioma formation) and contribute a great deal to biosafety. Because of the local application, we anticipated that systemic effects of anakinra, VEGF and PDGF would be negligible and tested this hypothesis using a pharmacokinetic model. This strategy of treatment could prevent the development of chronic heart failure, which is one of the most common complications after myocardial infarction.</p>
 +
<h2>Results</h2>
 +
<h3>Pharmacokinetic modeling of the distribution of anakinra in the local anti-inflammatory therapy</h3>
 +
<p>We prepared a pharmacokinetic model for the local therapy of ischaemic heart disease with anakinra. This model (described in details in the <a href="https://2012.igem.org/Team:Slovenia/Modeling">Modeling</a> section) demonstrates achievement of the local therapeutic concentrations of anakinra, while the systemic concentration remains negligible in contrast to systemic application of anti-inflammatory therapy. The serum half life of anakinra is very low so it needs to be administered daily for systemic application, but implementation of encapsulated cells with a constant production rate of anakinra would circumvent this.</p>
 +
 +
<p>
 +
<!-- figure 3 -->
 +
<table class="invisible" style="width:70%;  text-align:justify;" >
 +
<tbody  class="invisible">
 +
<tr class="invisible">
 +
<td class="invisible">
 +
<img  class="invisible" style="width:100%; height:auto;" src="https://static.igem.org/mediawiki/2012/1/1b/Svn12_anakinra_local.png"/>
 +
</td>
 +
</tr>
 +
<tr class="invisible"><td class="invisible">
 +
<b>Figure 3. Distribution of anakinra in different tissues and organs based on the pharmacokinetic model of the local therapy of ischaemic heart disease by microencapsulated engineered cells.</b> More intense (saturated) red colors indicate higher concentrations, while low concentrations are closer to transparent/white colors. The model demonstrates that anakinra is highly localized at the place of administration of microcapsules.
 +
</td></tr>
 +
</tbody>
 +
</table>
 +
</p>
 +
 +
 +
<h3>Detection of biological activity of produced anakinra</h3>
 +
<p>IL-1β signals through binding of IL-1 receptor type 1 (IL-1R1) and IL-1 receptor accessory protein (IL-1RAcP), which leads to a juxtaposition of the intracellular TIR domains of IL-1R1 and IL-1RAcP. The activation of the receptor complex triggers intracellular signaling which results in the activation of the transcription factor NF-κB and the mitogen-activated protein kinase (MAPK) pathways. Anakinra (IL-1 receptor antagonist; IL-1Ra) is a competitive inhibitor that prevents activation of the IL-1 receptor complex (Wang et al., 2010).</p>
 +
<p>We wanted to test the biological activity of anakinra produced in HEK293T. We designed an experiment where the inhibition of IL-1β signaling by anakinra was observed. HEK293T cells were transfected with NF-κB-inducible firefly luciferase reporter plasmid and constitutive <i>Renilla</i> luciferase plasmid for normalization. Transfected cells were preincubated with supernatant from anakinra-producing cells or with recombinant anakinra (rIL-1Ra). These cells were then stimulated with IL-1β and inhibition of NF-κB signaling pathway by anakinra was observed with double luciferase assay (Figure 4). Results show almost complete inhibition of IL-1β signaling with recombinant anakinra as well as with supernatant from anakinra-producing cells.</p>
 +
 +
 +
<img style="width:70%;" src="https://static.igem.org/mediawiki/2012/0/0a/Svn12_implementation_ischemia_fig4.png"></img>
 +
<center><p  style="width:68%;"><b>Figure 4. Inhibition of IL-1β signaling by anakinra.</b> HEK293T cells, transfected with reporter plasmid, were preincubated with supernatant of HEK293T cells producing anakinra under the control of a constitutive promoter or with recombinant anakinra (rIL-1Ra) (500 ng/mL). These cells were then stimulated with IL-1β (10 ng/mL) and inhibition of NF-κB signaling pathway by anakinra was observed with double luciferase assay.</p></center>
 +
 +
<p>We have calculated the therapeutic concentration of anakinra to be 25 ng/mL, which we derived from the anakinra IC_50 value (Dahlén et al., 2008). Our pharmacokinetic model of microencapsulated cell therapy predicted we would need production of approximately <b>30 µg per day in the target tissue</b>. Considering that in our experiments each cell produced around <b>1*10^-6 µg per day</b> and that the number of cells per capsule is 3000-15000 we calculated that we would need to implant between <b>2000-9000</b> capsules into a patch of ischaemic tissue, which would represent a volume of <b>15 to 75µL.</b> These estimates are very rough and would have to be tested experimentally in the tissue but provide the basis for judging the feasibility of this approach. Additional indication may also be provided by the successful application of the local gene therapy of myocarditis (Lim et al., 2002, Suzuki et al., 2001)</p>
<h2 style="color:grey;">References</h2>
<h2 style="color:grey;">References</h2>
<p style="color:grey;">
<p style="color:grey;">
-
Feld, J.J. and Hoofnagle, H. (2005) Mechanism of action of interferon and ribavirin in treatment of hepatitis C. <i>Nature</i> <b>436</b>, 967-72. <br/><br/>
+
Abbate, A., Salloum, F.N., Vecile, E., Das, A., Hoke, N.N., Straino, S., Biondi-Zoccai, G.G., Houser, J.E., Qureshi, I.Z., Ownby, E.D., Gustini, E., Biasucci, L.M., Severino, A., Capogrossi, M.C., Vetrovec, G.W., Crea, F., Baldi, A., Kukreja, R.C., and Dobrina, A. (2008) Anakinra, a recombinant human Interleukin-1 receptor antagonist, inhibits apoptosis in experimental acute myocardial infarction. <i>Circulation</i> <b>17</b>, 2670-2683. <br/><br/>
-
Loignon M, Perret S, Kelly J, Boulais D, Cass B, Bisson L, Afkhamizarreh F, Durocher Y. (2008) Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells. <i>BMC Biotechnol.</i> <b>8</b>, 65. <br/><br/>
+
 
-
Nakamura, T., Sakai, K., Nakamura, T. and Matsumoto, K. (2011) Hepatocyte growth factor twenty years on: Much more than a growth factor. <i>J Gastroenterol.  Hepatol.</i> <b>26</b>, 188-202.<br/><br/>
+
 
-
Oyagi, S., Hirose, M., Kojima, M., Okuyama, M., Kawase, M., Nakamura, T., Ohgushi, H. and Yagi, K. (2006) Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CCl4-injured rats. <i>J Hepatol.</i> <b>44</b>, 742-8.<br/><br/>
+
 
-
Pawlotsky, J.M. (2004) Pathophysiology of hepatitis C virus infection and related liver disease. <i>Trends in Microbiology.</i> <b>12</b>, 96-102.<br/><br/>
+
Banfi, A., von Degenfeld, G., Gianni-Barrera, R., Reginato, S., Merchant, M.J., McDonald, D.M., and Blau, H.M. (2012) Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB. <i>FASEB J.</i> <b>26</b>, 2486-2497. <br/><br/>
-
Ueki, T., Kaneda, Y., Tsutsui, H., Nakanishi, K., Sawa, Y., Morishita, R., Matsumoto, K., Nakamura, T., Takahashi, H., Okamoto, E. and Fujimoto, J. (1999) Hepatocyte growth factor gene therapy of liver cirrhosis in rats. <i>Nat. Medicine</i> <b>5</b>, 226-30.
+
 
 +
Dahlén, E., Barchan, K., Herrlander, D., Höjman, P., Karlsson, M., Ljung, L., Andersson, M., et al. 2008. Development of Interleukin-1 Receptor Antagonist Mutants with Enhanced Antagonistic Activity In Vitro and Improved Therapeutic Efficacy in Collagen-Induced Arthritis.<i>J Immunotoxicol</i> <b>5</b>, 189-99. <br/><br/>
 +
 
 +
Grothusen, C., Hagemann, A., Attmann, T., Braesen, J., Broch, O., Cremer ,J., and Schoettler, J. (2012) Impact of an interleukin-1 receptor antagonist and erythropoietin onexperimental myocardial ischemia/reperfusion injury. <i>Scientific World Journal</i> 737585. Epub 2012 May 2<br/><br/>
 +
 
 +
Lim BK, Choe SC, Shin JO, Ho SH, Kim JM, Yu SS, Kim S, Jeon ES. (2002) Local expression of interleukin-1 receptor antagonist by plasmid DNA improves mortality and decreases myocardial inflammation in experimental coxsackieviral myocarditis.<i>Circulation</i> <b>105</b>, 1278-81.<br/><br/>
 +
 
 +
Menzin, J., Wygant, G., Hauch, O., Jackel, J., and Friedman, M. (2008) One-year costs of ischemic heart disease among patients with acute coronary syndromes: findings from a multi-employer claims database. <i>Curr. Med. Res. Opin.</i> <b>24</b>, 461-468.<br/><br/>
 +
 
 +
Suzuki K, Murtuza B, Smolenski RT, Sammut IA, Suzuki N, Kaneda Y, Yacoub MH. (2001) Overexpression of interleukin-1 receptor antagonist provides cardioprotection against ischemia-reperfusion injury associated with reduction in apoptosis. <i>Circulation</i> <b>104</b>, I308-I3.<br/><br/>
 +
 
 +
Wang D., Zhang S., Li L., Xi Liu, Mei K. and Wang X. (2010) Structural insights into the assembly and activation of IL-1b with its receptors. <i>Nat. immunol</i> <b>11</b>, 10, 905-912.<br/><br/>
 +
Zhang, H., Zhu, S.J., Wang, W., Wei, Y.J., and Hu, S.S. (2008) Transplantation of microencapsulated genetically modified xenogeneic cells augments angiogenesis and improves heart function. <i>Gene Ther.</i> <b>15</b>, 40-48.
</p>
</p>
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Next: <a href="https://2012.igem.org/Team:Slovenia/ImplementationImpact">Impact >></a>
</b>
</b>

Latest revision as of 20:50, 26 October 2012


Ischaemic heart disease

We designed a device for the therapy of myocardial ischaemia, composed of microencapsulated mammalian cells that include a genetic bistable toggle switch with a positive feedback loop, where in the first state cells produce anakinra as the anti-inflammatory effector and in the second state they produce a stoichiometric amount of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor B (PDGF-BB) to promote angiogenesis in the damaged tissue.

A pharmacokinetic model demonstrated that implantation of the device into the injured heart tissue results in a high level of anakinra concentration within the affected tissue, while the systemic level of anakinra is negligible, preventing systemic immunosuppression.

We demonstrated that the production level of anakinra by engineered cells is sufficient for the therapeutic implementation of microencapsulated cells for this indication.


Ischaemic heart disease

Ischaemic heart disease is characterized by a reduced blood supply to the heart muscle, usually due to coronary artery disease (atherosclerosis of the coronary arteries). Ischaemic heart disease (which includes myocardial infarction, angina pectoris and heart failure when preceded by myocardial infarction) is the leading cause of mortality in most Western countries. Menzin et al. reported a total first-year cost average of 32,345 $ and as much as 61% of these costs were due to rehospitalisation (Menzin et al., 2008). Furthermore, survivors of acute myocardial infarction remain at high risk of death in the years after the event (Grothusen et al., 2012).

Current therapy and research

Acute myocardial infarction due to total occlusion of a coronary artery is treated with early reperfusion strategies to minimize the developing ischaemic myocardial damage. Current guidelines recommend interventional treatment via percutaneous transluminal coronary angioplasty (PTCA) of the occluded coronary artery followed by a stent implantation in the setting of acute myocardial infarction. In case of PTCA contraindications, operative revascularization therapy CABG (coronary artery bypass graft surgery) is recommended. However, many studies have shown a negative effect of reperfusion, resulting in additional myocardial injury due to activation of an inflammatory response (Grothusen et al., 2012; Abbate et al., 2008).

Interleukin-1a and b are one of the most important inflammatory cytokines which are responsible for leukocyte chemotaxis, macrophage activation, reactive oxygen species formation, endothelial dysfunction, and cardiomyocyte apoptosis. Thus, inhibition of IL-1 receptor complex activation can present a new important treatment target (Grothusen et al., 2012). Furthermore, recent studies have shown the cardioprotective properties of anakinra, which is a nonglycosylated, human recombinant competitive inhibitor of IL-1a and b signaling through binding to the IL-1 receptor complex (Abbate et al., 2008).

There is also experimental evidence of the important regenerating role of vascular endothelial growth factor (VEGF) after acute myocardial infarction (Banfi et al., 2012; Zhang et al., 2008). After binding to its receptor VEGFR1 and VEGFR2 it supports angiogenesis, inhibits endothelial cell apoptosis, promotes endothelial cell proliferation, and restores heart function. Although therapeutic angiogenesis by delivery of vascular growth factors is an attractive strategy, many clinical trials have thus far failed to show efficiency. One of the most likely explanations for this discrepancy is that VEGF induces growth of dysfunctional vessels, if expressed outside of a narrow dosage window (Banfi et al., 2012). Banfi et al. confirmed the hypothesis that co-delivery of platelet-derived growth factor-BB (PDGF-BB), which recruits pericytes, induced normal angiogenesis in skeletal muscle irrespective of VEGF levels. It was also shown that coexpression of VEGF and PDGF-BB encoded by separate vectors in different cells or in the same cells only partially corrected aberrant angiogenesis. In marked contrast, coexpression of both factors in every cell at a fixed relative level via a single bicistronic vector led to robust, uniformly normal angiogenesis, even when VEGF expression was high and heterogeneous. Secondly, a challenge has been that with the conventional gene transfer vectors, the growth factor concentration in target tissues had not reached sufficient levels or had not persisted long enough for triggering relevant vascular growth (Zhang et al., 2008). Cell transplantation strategies emerged as a promising approach to overcome this issue.

Zhang et al. reported the first transplantation of microencapsulated engineered xenogeneic CHO cells in post-infarction myocardium, which showed that the supplementation of VEGF from implanted xenogeneic cells could foster the formation of arterial collaterals, improve myocardial perfusion and thus also promote the regeneration of damaged myocardium augmented angiogenesis and improved heart function (Zhang et al., 2008).

Implementation of the microencapsulated engineered cellular device for the therapy of ischaemic heart disease

With our biological delivery system for biopharmaceuticals, we aim to improve the therapy of ischaemic heart disease by delivering multiple biopharmaceuticals in different time periods controlled by a physician. The encapsulated cells would be locally administered with intracardial injections via ventriculography, or intraoperatively in the proximity of the myocardial damage (Figure 1). In the first phase, after myocardial damage has occurred and reperfusion injury is expected, production of anakinra could halt the inflammatory response. In the second phase, the therapy would switch to the production of VEGF and PDGF-BB at stoichiometric concentrations, which could enhance the production of normal, robust vessels (Figure 2). After the therapeutic effect is reached, we could discontinue the synthesis of growth factors through the ability to control the viability of encapsulated allogenic cells. This could prevent the negative side effects of over-production of vascular growth factors (haemangioma formation) and contribute a great deal to biosafety. Because of the local application, we anticipated that systemic effects of anakinra, VEGF and PDGF would be negligible and tested this hypothesis using a pharmacokinetic model. This strategy of treatment could prevent the development of chronic heart failure, which is one of the most common complications after myocardial infarction.

Results

Pharmacokinetic modeling of the distribution of anakinra in the local anti-inflammatory therapy

We prepared a pharmacokinetic model for the local therapy of ischaemic heart disease with anakinra. This model (described in details in the Modeling section) demonstrates achievement of the local therapeutic concentrations of anakinra, while the systemic concentration remains negligible in contrast to systemic application of anti-inflammatory therapy. The serum half life of anakinra is very low so it needs to be administered daily for systemic application, but implementation of encapsulated cells with a constant production rate of anakinra would circumvent this.

Detection of biological activity of produced anakinra

IL-1β signals through binding of IL-1 receptor type 1 (IL-1R1) and IL-1 receptor accessory protein (IL-1RAcP), which leads to a juxtaposition of the intracellular TIR domains of IL-1R1 and IL-1RAcP. The activation of the receptor complex triggers intracellular signaling which results in the activation of the transcription factor NF-κB and the mitogen-activated protein kinase (MAPK) pathways. Anakinra (IL-1 receptor antagonist; IL-1Ra) is a competitive inhibitor that prevents activation of the IL-1 receptor complex (Wang et al., 2010).

We wanted to test the biological activity of anakinra produced in HEK293T. We designed an experiment where the inhibition of IL-1β signaling by anakinra was observed. HEK293T cells were transfected with NF-κB-inducible firefly luciferase reporter plasmid and constitutive Renilla luciferase plasmid for normalization. Transfected cells were preincubated with supernatant from anakinra-producing cells or with recombinant anakinra (rIL-1Ra). These cells were then stimulated with IL-1β and inhibition of NF-κB signaling pathway by anakinra was observed with double luciferase assay (Figure 4). Results show almost complete inhibition of IL-1β signaling with recombinant anakinra as well as with supernatant from anakinra-producing cells.

Figure 4. Inhibition of IL-1β signaling by anakinra. HEK293T cells, transfected with reporter plasmid, were preincubated with supernatant of HEK293T cells producing anakinra under the control of a constitutive promoter or with recombinant anakinra (rIL-1Ra) (500 ng/mL). These cells were then stimulated with IL-1β (10 ng/mL) and inhibition of NF-κB signaling pathway by anakinra was observed with double luciferase assay.

We have calculated the therapeutic concentration of anakinra to be 25 ng/mL, which we derived from the anakinra IC_50 value (Dahlén et al., 2008). Our pharmacokinetic model of microencapsulated cell therapy predicted we would need production of approximately 30 µg per day in the target tissue. Considering that in our experiments each cell produced around 1*10^-6 µg per day and that the number of cells per capsule is 3000-15000 we calculated that we would need to implant between 2000-9000 capsules into a patch of ischaemic tissue, which would represent a volume of 15 to 75µL. These estimates are very rough and would have to be tested experimentally in the tissue but provide the basis for judging the feasibility of this approach. Additional indication may also be provided by the successful application of the local gene therapy of myocarditis (Lim et al., 2002, Suzuki et al., 2001)

References

Abbate, A., Salloum, F.N., Vecile, E., Das, A., Hoke, N.N., Straino, S., Biondi-Zoccai, G.G., Houser, J.E., Qureshi, I.Z., Ownby, E.D., Gustini, E., Biasucci, L.M., Severino, A., Capogrossi, M.C., Vetrovec, G.W., Crea, F., Baldi, A., Kukreja, R.C., and Dobrina, A. (2008) Anakinra, a recombinant human Interleukin-1 receptor antagonist, inhibits apoptosis in experimental acute myocardial infarction. Circulation 17, 2670-2683.

Banfi, A., von Degenfeld, G., Gianni-Barrera, R., Reginato, S., Merchant, M.J., McDonald, D.M., and Blau, H.M. (2012) Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB. FASEB J. 26, 2486-2497.

Dahlén, E., Barchan, K., Herrlander, D., Höjman, P., Karlsson, M., Ljung, L., Andersson, M., et al. 2008. Development of Interleukin-1 Receptor Antagonist Mutants with Enhanced Antagonistic Activity In Vitro and Improved Therapeutic Efficacy in Collagen-Induced Arthritis.J Immunotoxicol 5, 189-99.

Grothusen, C., Hagemann, A., Attmann, T., Braesen, J., Broch, O., Cremer ,J., and Schoettler, J. (2012) Impact of an interleukin-1 receptor antagonist and erythropoietin onexperimental myocardial ischemia/reperfusion injury. Scientific World Journal 737585. Epub 2012 May 2

Lim BK, Choe SC, Shin JO, Ho SH, Kim JM, Yu SS, Kim S, Jeon ES. (2002) Local expression of interleukin-1 receptor antagonist by plasmid DNA improves mortality and decreases myocardial inflammation in experimental coxsackieviral myocarditis.Circulation 105, 1278-81.

Menzin, J., Wygant, G., Hauch, O., Jackel, J., and Friedman, M. (2008) One-year costs of ischemic heart disease among patients with acute coronary syndromes: findings from a multi-employer claims database. Curr. Med. Res. Opin. 24, 461-468.

Suzuki K, Murtuza B, Smolenski RT, Sammut IA, Suzuki N, Kaneda Y, Yacoub MH. (2001) Overexpression of interleukin-1 receptor antagonist provides cardioprotection against ischemia-reperfusion injury associated with reduction in apoptosis. Circulation 104, I308-I3.

Wang D., Zhang S., Li L., Xi Liu, Mei K. and Wang X. (2010) Structural insights into the assembly and activation of IL-1b with its receptors. Nat. immunol 11, 10, 905-912.

Zhang, H., Zhu, S.J., Wang, W., Wei, Y.J., and Hu, S.S. (2008) Transplantation of microencapsulated genetically modified xenogeneic cells augments angiogenesis and improves heart function. Gene Ther. 15, 40-48.


Next: Impact >>