Team:Slovenia

From 2012.igem.org

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Revision as of 21:37, 24 September 2012

The Challenge

Biological drugs such as hormones, enzymes, cytokines or antibodies are increasingly used to treat different diseases. Due to systemic administration, these drugs often have adverse effects. Additionally, the high cost of biopharmaceutials imposes a heavy burden on health systems.

We aimed to develop a safe and cost-effective biological in situ production and delivery system for biological drugs to increase the quality of patients' lives. This system should increase compliance to the therapy, minimize the number of required invasive procedures and introduce advanced multistage therapy while local administration could reduce the side effects.

We addressed this challenge by implementing microencapsulated engineered mammalian cells that can be regulated from the outside to produce different therapeutics, should be reliable, cost effective and safe.

Project Results

The switch We designed a new type of bistable toggle switch for mammalian cells based on designed DNA-binding proteins to enable simultaneous introduction of several orthogonal switches and construction of complex logic devices. We discovered that a classical toggle switch topology was ineffective if based on TAL effectors, because they bind to DNA non-cooperatively as monomers. We solved this problem by designing a switch comprised of a pair of mutual repressors (TAL-KRAB) coupled with a pair of activators (TAL-VP16) that form a positive feedback loop. This arrangement resulted in experimentally confirmed bistability in mammalian cells that can be regulated by small molecule inducers. Read more...	Safety In order to enable safe application of engineered cells for the microencapsulation based therapy we designed safety mechanisms to degrade the alginate capsules at the end of the therapy, terminate therapeutic cells by induction of apoptosis and introduction of an escape tag that marks any escaped cells for elimination by natural killer cells of the host. Read more...	Implementation We implemented the effector therapeutics for therapy of hepatitis C and ischaemic heart disease by introducing five different therapeutic proteins that could, in agreement with our pharmacokinetic models, reduce the side effects and improve the efficiency of the therapy. Switching between production of effectors with antiviral or anti-inflammatory effect and tissue regeneration could be regulated by a physician by delivery of small molecule inducers from the outside. Read more...
Modeling Exhaustive mathematical modeling demonstrated that the classical toggle genetic switch arrangement is not stable without cooperativity. At the same time, the modeling confirmed an improved robustness of the switch when using modular DNA binding element-based transcriptional regulators that formed two positive feedback loops. This topology does not require cooperativity since nonlinearity is introduced by the positive feedback loop. A pharmacokinetic model of the local delivery of therapeutics by microencapsulated cells predicted that this type of drug delivery should have reduced systemic side effects. Read more...	Society Different aspects of our project as well as the medical applications of synthetic biology were discussed with a wide range of stakeholders, including medical professionals, patients, regulators, general public, media and scientists that will have to participate in the succesfull introduction of synthetic biology applications in clinical use. We engaged a network of high schools to introduce younger generations to synthetic biology and its use in medicine as well as the broad range of its other potential uses. Read more...	Perspectives The safety device, including the termination, escape killing and degradation components will allow implementation for different therapeutic purposes, we plan to establish stable cell lines, each containing an integrated switch with the selected therapeutic effectors and safety mechanisms, the designed orthogonal TAL-based transcriptional factor logic will allow construction of tens or hundreds of different switches and achievement of multistable states and other complex logical devices, We plan to initiate the first in vivo tests on ischaemia and wound healing. Read more...

Abstract in plain english

Biological drugs have lately been replacing chemical drugs in the treatement of numerous diseases due to their more specific nature. The current route of biological drug administration is usually intravenous or subcutaneous, which means that the drug is distributed more or less throughout the whole body, although its function should often be restricted to a specific organ or tissue. This can cause serious side effects, requires larger dosage and consequently raises the price of therapy. Our solution of this problem is to implant cells producing biological drugs inside the very tissue where the drug is required. The drug producing cells are safely sealed inside microcapsules, preventing dissemination of these cells and protecting them from destruction by the cells of the host immune system. The implanted cells are able to produce different types of drugs and switching between them can be controlled by a physician depending on the stage of the disease. We invented a new type of switch that will allow selecting production of different biological drug combinations in the affected tissue. We have modified cells for use in the therapy of hepatitis C, where the engineered cells produce a biological drug that has antiviral activity followed by a drug that improves liver regeneration. For the therapy after a heart attack we designed cells to suppress inflammation and promote the formation of new blood vessels around the affected tissue. A medical doctor may initiate self-destruction of the cells and capsules by an outside stimulus when the therapy is over or at any other given time. We believe our system to be safe and effective and applicable to the therapy of different types of diseases.

Outcome

Achievements in technical details:

this is the first experimental implementation of a bistable toggle switch based on noncooperative DNA-binding proteins and the first bistable switch based on designed DNA-binding proteins,
we designed and tested a bistable toggle switch for mammalian cells based on orthogonal TAL-repressors and activators,
modeling demonstrated improved robustness of a switch based on a positive feedback loop, with respect to leaky transcription,
we introduced three safety mechanisms into microencapsulated mammalian cells:
- an escape tag for cell elimination by natural killer cells,
- a secretory alginate lyase for degradation of alginate microcapsules,
- induction of apoptosis of therapeutic cells by the introduction of a prodrug,
we introduced interferon alpha/hepatocyte growth factor as an effector pair for the therapy of hepatitis C to inactivate the virus followed by the promotion of liver regeneration,
we introduced anakinra/vascular endothelial growth factor:platelet-derived growth factor as effectors for therapy of ischaemia to suppress inflammation followed by angiogenesis.
we deposited 90 BioBricks to the Registry, most of them used in functional devices,
we improved an existing BioBrick.

Project sponsors

@@ Line 398: / Line 398: @@
 <tr class="summary">
 <td class="summary" style="font-size:110%;">
-<strong style="font-size:120%;">Challenge</strong><br/>
+<strong style="font-size:120%;">The Challenge</strong><br/>
 <br />
-<p>Biological drugs are very effective and
+<p>Biological drugs such as hormones, enzymes, cytokines or antibodies are increasingly used to treat different diseases. Due to systemic administration, these drugs often have adverse effects. Additionally, the high cost of biopharmaceutials imposes a heavy burden on health systems.</p>
-are increasingly used to treat different diseases. Often, due to their systemic administration,
+<p>We aimed to develop a safe and cost-effective biological in situ production and delivery system for biological drugs to increase the quality of patients' lives. This system should increase compliance to the therapy, minimize the number of required invasive procedures and introduce advanced multistage therapy while local administration could reduce the side effects. </p>
-adverse effects are observed. Additionally high cost of biopharmaceutials imposes a great burden
+<p>We addressed this challenge by implementing microencapsulated engineered mammalian cells that can be regulated from the outside to produce different therapeutics, should be reliable, cost effective and safe.</p>
-on health systems. We aimed to develop a safe and cost-effective biological delivery system for
-biopharmaceuticals, which would increase the quality of patients' lives. This system would
-increase compliance to the therapy, minimize the number of required invasive procedures,
-introduce more effective multistage therapy while the local administration will reduce the side-effects. </p>
-<p>We addressed this challenge by implementing microencapsulated engineered mammalian cells
-that can be regulated from the outside to produce different therapeutics and comprise safety mechanisms.</p>
 </td>
 </tr>
@@ Line 414: / Line 408: @@
 <!-- /summary table -->
+<br />
+<br />
+<strong style="font-size:120%;">Project Results</strong><br/>
+<br />
 <!-- splash table -->
@@ Line 424: / Line 422: @@
 <div class="ttip" style="top:100px;">
 <strong>The switch</strong>
-<p>We designed a <strong>new type of bistable toggle switch for mammalian cells</strong>
+<p>We <b>designed a new type of bistable toggle switch for mammalian cells</b> based on designed DNA-binding proteins to enable simultaneous introduction of several orthogonal switches and construction of complex logic devices. We discovered that a <b>classical toggle switch topology was ineffective</b> if based on TAL effectors, because they bind to DNA non-cooperatively as monomers. We solved this problem by designing a switch comprised of a pair of mutual repressors (TAL-KRAB) coupled with a pair of activators (TAL-VP16) that form a <b>positive feedback loop</b>. This arrangement resulted in experimentally confirmed bistability in mammalian cells that can be regulated by small molecule inducers. <a href="https://2012.igem.org/Team:Slovenia/TheSwitch">Read more...</a></p>
-based on designed DNA-binding proteins, which would allow the simultaneous introduction of
-several orthogonal switches and construction of complex logic devices.
-We discovered that the <strong>classical toggle switch topology was ineffective</strong> since TAL effectors bind
-noncooperatively as monomers. We solved this problem by designing a switch comprising a pair of
-mutual repressors (TAL-KRAB) coupled with a pair of activators (TAL-VP16) that form a <strong>positive feedback loop</strong>.
-This arrangement resulted in experimental confirmation of bistability in mammalian cells that can be regulated by
-small molecule inducers. <a href="https://2012.igem.org/Team:Slovenia/TheSwitch">Read more...</a></p>
 </div>
 </td>
@@ Line 439: / Line 430: @@
 <div class="ttip" style="top:100px;">
 <strong>Safety</strong>
-<p>We tailored the benefits of microencapsulated engineered cells by designing safety mechanisms
+<p>In order to enable safe application of engineered cells for the microencapsulation based therapy we designed safety mechanisms to <b>degrade the alginate capsules</b> at the end of the therapy, terminate therapeutic cells by induction of apoptosis and introduction of an escape tag that <b>marks any escaped cells for elimination</b> by natural killer cells of the host. <a href="https://2012.igem.org/Team:Slovenia/SafetyMechanisms">Read more...</a></p>
- to degrade the alginate capsules at the end of therapy,
- terminate therapeutic cells by induction of apoptosis and introduction of an escape killing tag
- that marks potential escaped cells to destruction by the host natural killer cells. <a href="https://2012.igem.org/Team:Slovenia/SafetyMechanisms">Read more...</a></p>
 </div>
 </td>
@@ Line 450: / Line 438: @@
 <div class="ttip" style="top:100px;">
 <strong>Implementation</strong>
-<p>We implemented the effector therapeutics for therapy of hepatitis C
+<p>We implemented the effector therapeutics for therapy of hepatitis C and ischaemic heart disease by introducing five different therapeutic proteins that could, in agreement with our pharmacokinetic models, reduce the side effects and improve the efficiency of the therapy. Switching between production of effectors with antiviral or anti-inflammatory effect and tissue regeneration could be regulated by a physician by delivery of small molecule inducers from the outside.  <a href="https://2012.igem.org/Team:Slovenia/Implementation">Read more...</a></p>
-and ischaemic heart disease, by introducing five different therapeutic proteins that could,
-in agreement with our pharmacokinetic models, reduce the side effects and improve the efficiency of the therapy.
- Switching between production of effectors with antiviral or anti-inflammatory effect and tissue regeneration
- could be regulated by the physician by delivery of small molecule inducers from the outside.  <a href="https://2012.igem.org/Team:Slovenia/Implementation">Read more...</a></p>
 </div>
 </td>
@@ Line 466: / Line 450: @@
 <div class="ttip" style="top:400px;">
 <strong>Modeling</strong>
-<p>Exhaustive modeling demonstrated that the classical toggle switch is not stable without cooperativity
+<p>Exhaustive mathematical modeling demonstrated that the classical toggle genetic switch arrangement is not stable without cooperativity. At the same time, the modeling confirmed an improved robustness of the switch when using modular DNA binding element-based transcriptional regulators that formed two positive feedback loops. This topology does not require cooperativity since nonlinearity is introduced by the positive feedback loop. A pharmacokinetic model of the local delivery of therapeutics by microencapsulated cells predicted that this type of drug delivery should have reduced systemic side effects. <a href="https://2012.igem.org/Team:Slovenia/Modeling">Read more...</a></p>
-while it confirmed the improved robustness of the switch that included two positive feedback loops.
- This topology does not require cooperativity since the nonlinearity is introduced by positive feedback loop.
- Pharmacokinetic model of the local delivery of therapeutics by microencapsulated cells predicted that this system
- has reduced systemic side effects. <a href="https://2012.igem.org/Team:Slovenia/Modeling">Read more...</a></p>
 </div>
 </td>
@@ Line 478: / Line 458: @@
 <div class="ttip" style="top:400px;">
 <strong>Society</strong>
-<p>Different aspects of the project as well as medical applications of
+<p>Different aspects of our project as well as the medical applications of synthetic biology were discussed with a wide range of stakeholders, including medical professionals, patients, regulators, general public, media and scientists that will have to participate in the succesfull introduction of synthetic biology applications in clinical use. We engaged a network of high schools to introduce younger generations to synthetic biology and its use in medicine as well as the broad range of its other potential uses.  <a href="https://2012.igem.org/Team:Slovenia/Society">Read more...</a></p>
-synthetic biology were discussed with a wide range of stakeholders,
-including medical professionals, patients, regulators, general public and
-scientists that will support introduction of this technology into clinical use.
- We involved a network of high school students into the dissemination. <a href="https://2012.igem.org/Team:Slovenia/Society">Read more...</a></p>
 </div>
 </td>
@@ Line 491: / Line 467: @@
 <strong>Perspectives</strong>
 <ul style="padding-left:30px;">
-<li>the safety device including termination, escape and degradation component will allow implementation for different therapeutic purposes,</li>
+<li> The safety device, including the termination, escape killing and degradation components will allow implementation for different therapeutic purposes,</li>
-<li>stable cell lines comprising switch with the selected therapeutic effectors and safety mechanisms will be established,</li>
+<li>we plan to establish stable cell lines, each containing an integrated switch with the selected therapeutic effectors and safety mechanisms,</li>
-<li>we plan to initiate in vivo tests first on ischaemia and wound healing,</li>
+<li>the designed orthogonal TAL-based transcriptional factor logic will allow construction of tens or hundreds of different switches and achievement of multistable states and other complex logical devices,</li>
-<li>the designed orthogonal TAL-transcriptional factor logics will allow introduction multistability from several parallel switches and other complex logical devices.</li>
+<li>We plan to initiate the first in vivo tests on ischaemia and wound healing.</li>
 </ul>
 <p><a href="https://2012.igem.org/Team:Slovenia/SocietyEthics">Read more...</a></p>
@@ Line 511: / Line 487: @@
 <tr class="summary">
 <td class="summary" style="font-size:110%;">
-<strong style="font-size:120%;">Abstract for non-scientists</strong><br/>
+<strong style="font-size:120%;">Abstract in plain english</strong><br/>
 <br />
-<p>We designed a new type of therapy where we modify human cells,
+<p>Biological drugs have lately been replacing chemical drugs in the treatement of numerous diseases due to their more specific nature. The current route of biological drug administration is usually intravenous or subcutaneous, which means that the drug is distributed more or less throughout the whole body, although its function should often be restricted to a specific organ or tissue. This can cause serious side effects, requires larger dosage and consequently raises the price of therapy. Our solution of this problem is to implant cells producing biological drugs inside the very tissue where the drug is required. The drug producing cells are safely sealed inside microcapsules, preventing dissemination of these cells and protecting them from destruction by the cells of the host immune system. The implanted cells are able to produce different types of drugs and switching between them can be controlled by a physician depending on the stage of the disease.  We invented a new type of switch that will allow selecting production of different biological drug combinations in the affected tissue. We have modified cells for use in the therapy of hepatitis C, where the engineered cells produce a biological drug that has antiviral activity followed by a drug that improves liver regeneration. For the therapy after a heart attack we designed cells to suppress inflammation and promote the formation of new blood vessels around the affected tissue. A medical doctor may initiate self-destruction of the cells and capsules by an outside stimulus when the therapy is over or at any other given time. We believe our system to be safe and effective and applicable to the therapy of different types of diseases.<p>
-pack them into small capsules and introduce into the diseased tissue.
-In order to make this therapy useful we invented new type of switches that will allow
-the medical doctors to turn on or off production of different biological drug in the patient.
- At the end of the therapy the capsule will be degraded and cells that produced drugs will be
- destroyed. For the first applications we selected therapy of hepatitis C, where we induce production
- of drug that has antiviral activity and at the later stage a drug that helps regeneration of liver.
- For the therapy of heart infarction we designed cells that suppress inflammation and promote formation
- of new blood vessels around the affected tissue. In our experiments we demonstrated function of new
- devices that have to be integrated into final therapy. Students also made mathematical model of switch
- and on the distribution of drugs throughout body that should decrease the side effects of therapy.</p>
 </td>
@@ Line 546: / Line 512: @@
 <strong>Achievements in technical details:</strong>
 <ul style="padding-left: 30px;">
-<li>this is the first experimental implementation of a bistable toggle switch based on noncooperative DNA-binding elements and the first bistable switch based on designed DNA-binding elements,</li>
+<li> this is the first experimental implementation of a bistable toggle switch based on noncooperative DNA-binding proteins and the first bistable switch based on designed DNA-binding proteins,</li>
-<li>we designed and tested bistable toggle switch for mammalian cells based on orthogonal TAL-repressors and activators,</li>
+<li>we designed and tested a bistable toggle switch for mammalian cells based on orthogonal TAL-repressors and activators,</li>
-<li>modeling demonstrated improved robustness of a switch based on a positive feedback loop with respect to leaky transcription,</li>
+<li> modeling demonstrated improved robustness of a switch based on a positive feedback loop, with respect to leaky transcription,</li>
-<li>introduced three safety mechanisms into microencapsulated mammalian cells:
+<li>we introduced three safety mechanisms into microencapsulated mammalian cells:
 	<ul class="circle" style="padding-left: 50px;">
-	<li>escape tag for cell elimination by natural killer cells,</li>
+	<li>an escape tag for cell elimination by natural killer cells,</li>
-	<li>secretory alginate lyase for degradation of alginate microcapsules,</li>
+	<li>a secretory alginate lyase for degradation of alginate microcapsules,</li>
 	<li>induction of apoptosis of therapeutic cells by the introduction of a prodrug,</li>
 	</ul>
 	</li>
-<li>introduced IFN-alpha/HGH effector pair for the therapy of hepatitis C to inactivate the virus and promote liver regeneration in the later stage,</li>
+<li>we introduced interferon alpha/hepatocyte growth factor as an effector pair for the therapy of hepatitis C to inactivate the virus followed by the promotion of liver regeneration, </li>
-<li>introduced anakinra/VEGF-PDGF-B for therapy of ischaemia to suppress inflammation and promote angiogenesis in the later stage,</li>
+<li>  we introduced anakinra/vascular endothelial growth factor:platelet-derived growth factor as effectors for therapy of ischaemia to suppress inflammation followed by angiogenesis.</li>
-<li>deposited 89 BioBricks,</li>
+<li>we deposited 90 BioBricks to the Registry, most of them used in functional devices,</li>
-<li>improved an existing BioBrick.</li>
+<li>we improved an existing BioBrick.</li>
 </ul>
 </td>