Team:Freiburg Bioware/Project/Results

From 2010.igem.org

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<p>
<p>
Gene delivery is a very promising approach, which yet has to live up to its potentials. Despite several clinical trials, no modular and at the same time exhaustive line of attack has been published. We took advantage of current knowledge to generate a fully modular recombinant Adeno-associated virus (rAAV) based system, which incorporates an extensive set of known surface modifications. In addition, we utilize recently developed binding molecules to generate and demonstrate a novel tumor-targeting approach for rAAV. Our modules have been first extensively tested individually and then in combination for viral production and infectivity. They compared well or exceeded performance of existing systems. The BioBrick-compatible viral vectors demonstrated their ability of tissue-specific delivery of genes coding for fluorescent protein reporters as well as prodrug-activating enzymes for tumor therapy. Using these viruses, we demonstrate specific prodrug-mediated killing of human tumor cells overexpressing a tumor-specific receptor while a reference cell line was unaffected.
Gene delivery is a very promising approach, which yet has to live up to its potentials. Despite several clinical trials, no modular and at the same time exhaustive line of attack has been published. We took advantage of current knowledge to generate a fully modular recombinant Adeno-associated virus (rAAV) based system, which incorporates an extensive set of known surface modifications. In addition, we utilize recently developed binding molecules to generate and demonstrate a novel tumor-targeting approach for rAAV. Our modules have been first extensively tested individually and then in combination for viral production and infectivity. They compared well or exceeded performance of existing systems. The BioBrick-compatible viral vectors demonstrated their ability of tissue-specific delivery of genes coding for fluorescent protein reporters as well as prodrug-activating enzymes for tumor therapy. Using these viruses, we demonstrate specific prodrug-mediated killing of human tumor cells overexpressing a tumor-specific receptor while a reference cell line was unaffected.
-
During the development of our system we mastered several hurdles and developed the know-how to manipulate the viral genome. We had to generate a new version of the standard iGEM backbone, since we need additional singular restriction sites to enable swapping of sequences coding for the viral loop structures, which can be used to determine the tropism. Our systems allows to exchange two loops of the virus for a His affinity tag, which enables purification, a biotinylation tag, or an antibody binding motif. We demonstrated functional assembly of viruses with all three modifications.
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</p>
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In addition we established two N-terminal fusions to the capsid proteins using the binding scaffolds of DARPins and Affibodies. To our knowledge, neither the use of these motifs in a viral setting nor the tumor targeting of rAAV by N-terminal fusion have been demonstrated so far.
+
<p>
 +
During the development of our system, we mastered several hurdles and developed the know-how to manipulate the viral genome. We had to generate a new version of the standard iGEM backbone, since we need additional singular restriction sites to enable swapping of sequences coding for the viral loop structures, which can be used to determine the tropism. Our systems allows for the exchange of two loops of the virus for either a His-tag, which enables purification, or a biotinylation-tag, or an antibody binding module. We demonstrated the functional assembly of viruses with all three modifications.
 +
</p>
 +
<p>
 +
In addition, we established two N-terminal fusions to the capsid proteins using the binding scaffolds of DARPins and Affibodies. To our knowledge, neither the use of these motifs in a viral setting nor the tumor targeting of rAAV by N-terminal fusion have been demonstrated so far.
 +
</p>
 +
<p>
We applied a wide range of techniques to achieve our goals. Cloning was performed in E. coli, viruses were produced in a human kidney cell line, and viral infections were tested in further human cell lines. Viruses were purified by liquid chromatography and virus samples were analyzed by quantitative real-time PCR and ELISA. Expression of virally delivered reporter genes was analyzed by fluorescence microscopy including time-lapse imaging. Expression and cell viability upon infection was also assessed by flow cytometry and MTT-assays. Viral samples were additionally analyzed by atomic force microscopy and electron microscopy.
We applied a wide range of techniques to achieve our goals. Cloning was performed in E. coli, viruses were produced in a human kidney cell line, and viral infections were tested in further human cell lines. Viruses were purified by liquid chromatography and virus samples were analyzed by quantitative real-time PCR and ELISA. Expression of virally delivered reporter genes was analyzed by fluorescence microscopy including time-lapse imaging. Expression and cell viability upon infection was also assessed by flow cytometry and MTT-assays. Viral samples were additionally analyzed by atomic force microscopy and electron microscopy.
-
We provide an extensive lab journal, a well documented set of over 100 BioBricks and a detailed manual for the Virus Construction Kit.
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</p>
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<p>
-
 
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Last but not least, we provide an extensive lab journal, a well documented set of over 100 BioBricks and a detailed manual for the Virus Construction Kit.
-
 
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</p>
-
List with milestones reaches
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 +
<br>
Our key achievements are:
Our key achievements are:
<ul>
<ul>
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<li>A BioBrick set and assembled plasmids for gene delivery.<br>
-
<li>A BioBrick set and assembled plasmids for gene delivery. This set comprises the AAV ITRs, a CMV and a hTERT promoter, as well as the genes of interest mVenus, mCherry as reporters and thymidine kinase and cytosin deaminase constructs for prodrug activation, as well as a beta-globin intron and a hGH polyadenylation tag to control gene expression.</li>
+
This set comprises the AAV ITRs, a CMV and a hTERT promoter, as well as the genes of interest mVenus, mCherry as reporters and thymidine kinase and cytosin deaminase constructs for prodrug activation, as well as a beta-globin intron and a hGH polyadenylation tag to control gene expression.</li>
-
<li>A Biobrick set and plasmids for viral capsid production and modification. This set comprises an Affibody and a DARPin targeting the EGF-receptor and a set of linkers which can be utilized for N-terminal fusions. A modified gene coding for viral capsid proteins, which is modified for the fusion. In addition we provide a viral capsid gene for loop modification and the fitting loop modifications coding for a His-tag, biotinylation sequence, and antibody binding site.</li>
+
<li>A Biobrick set and plasmids for viral capsid production and modification.<br>
 +
This set comprises an Affibody and a DARPin targeting the EGF-receptor and a set of linkers which can be utilized for N-terminal fusions. A modified gene coding for viral capsid proteins, which is modified for the fusion. In addition we provide a viral capsid gene for loop modification and the fitting loop modifications coding for a His-tag, biotinylation sequence, and antibody binding site.</li>
<li>An extensive set of experiments demonstrating the functionality of our constructs.</li>
<li>An extensive set of experiments demonstrating the functionality of our constructs.</li>
</ul>
</ul>
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<a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid" name="modularization" class="onlyAnchor"><h2>Modularization</h2></a>
<a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid" name="modularization" class="onlyAnchor"><h2>Modularization</h2></a>
<p>
<p>
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We converted a recombinant Adeno-associated virus to the BioBrick standard. This modularization is grouped into two parts: first, the generation of a gene of interest (vector) plasmid and second the generation of vector for the modified capsid proteins.
+
We converted a recombinant Adeno-associated virus to the BioBrick standard. This modularization is grouped into two parts: first, the generation of a gene of interest (vector) plasmid and second, the generation of vectors for the modified capsid proteins.
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<br />
+
</p>
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<b><a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">Gene of interest</a></b><br />
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<p>
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The Virus Construction Kit enables researchers to encapsidate virtually any given DNA sequence into AAV-2 particles. As one example from therapeutic focus of our project, prodrug-activating enzymes are provided within the kit for efficient tumor cell killing. Additionally included fluorescent proteins allow monitoring of transduced cells by fluorescence microscopy and flow cytometry. Transgene expression can be fine-tuned using promoters of different specificity and enhancer elements also provided.
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<b><a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">Gene of Interest (GOI) Plasmid</a></b><br />
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<br>
+
The Virus Construction Kit enables researchers to encapsidate virtually any given DNA sequence into AAV-2 particles. As one example from therapeutic focus of our project, prodrug-activating enzymes are provided within the kit for efficient tumor cell killing. Additionally included fluorescent proteins allow monitoring of transduced cells by fluorescence microscopy and flow cytometry. Transgene expression can be fine-tuned using promoters of different specificity and enhancer elements also provided. <a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">more</a>
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</p>
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<b><a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">Capsid</a></b><br />
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<p>
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AAV-2 genes essential for the production of viral particles in a specialized cell line were identified by literature search, isolated on genetic level and modified to meet the requirements of BioBrick assembly. For this purpose, a specialized variant of the iGEM default backbone was created, proven functional and submitted.
+
<b><a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">Capsid Plasmids</a></b><br />
 +
AAV-2 genes essential for the production of viral particles in a specialized cell line were identified by literature search, isolated on genetic level and modified to meet the requirements of BioBrick assembly. For this purpose, a specialized variant of the iGEM default backbone was created, proven functional and submitted. <a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">more</a><br />
</p>
</p>
</td>
</td>
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<td>
<a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Targeting_Fusion" name="targeting" class="onlyAnchor"><h2>Targeting</h2></a>
<a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Targeting_Fusion" name="targeting" class="onlyAnchor"><h2>Targeting</h2></a>
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<p>One aim of our research is on the one hand to knock down the natural tropism of the Adeno-associates virus particles and on the other hand to specifically target tumor cells. This is achieved by genetic engineering of the virus surface. For this purpose, two different strategies were developed, including <a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/insertion of motifs into surface-exposed loops">Targeting via Loops</a> or <a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Targeting_Fusion">fusion to the N-terminus of the viral protein VP2</a>.<br>
+
<p>Retargeting of the viral particles was achieved by knocking down the natural tropism of the Adeno-associates virus particles and at the same time introducing capsid modifications for specifically targeting tumor cells. For this purpose, two different strategies were developed, which were <a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/insertion of motifs into surface-exposed loops">targeting via loops</a> or <a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Targeting_Fusion">targeting via fusion to the N-terminus of the viral protein VP2</a>.<br>
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<br>
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</p>
</p>
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<td>
<table class="highlights">
<table class="highlights">
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<td>
<a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Arming" name="arming" class="onlyAnchor"><h2>Arming</h2></a>
<a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Arming" name="arming" class="onlyAnchor"><h2>Arming</h2></a>
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<p>The specifically targeted tumor cells were killed by prodrug activation approaches. Viral particles were charged with thymidine kinase and cytosine deaminase constructs to induce apoptosis in cancer cells upon delivery of ganciclovir or 5-Fluorocytosine, respectively.<a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Arming">[more]</a>
+
<p>The specifically targeted tumor cells were killed by prodrug activation approaches. Viral particles were charged with thymidine kinase or cytosine deaminase constructs to kill cancer cells upon delivery of ganciclovir or 5-Fluorocytosine, respectively. <a href="http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Arming">[more]</a>
</p>
</p>
</td>
</td>

Revision as of 13:27, 14 January 2011

Summary

Gene delivery is a very promising approach, which yet has to live up to its potentials. Despite several clinical trials, no modular and at the same time exhaustive line of attack has been published. We took advantage of current knowledge to generate a fully modular recombinant Adeno-associated virus (rAAV) based system, which incorporates an extensive set of known surface modifications. In addition, we utilize recently developed binding molecules to generate and demonstrate a novel tumor-targeting approach for rAAV. Our modules have been first extensively tested individually and then in combination for viral production and infectivity. They compared well or exceeded performance of existing systems. The BioBrick-compatible viral vectors demonstrated their ability of tissue-specific delivery of genes coding for fluorescent protein reporters as well as prodrug-activating enzymes for tumor therapy. Using these viruses, we demonstrate specific prodrug-mediated killing of human tumor cells overexpressing a tumor-specific receptor while a reference cell line was unaffected.

During the development of our system, we mastered several hurdles and developed the know-how to manipulate the viral genome. We had to generate a new version of the standard iGEM backbone, since we need additional singular restriction sites to enable swapping of sequences coding for the viral loop structures, which can be used to determine the tropism. Our systems allows for the exchange of two loops of the virus for either a His-tag, which enables purification, or a biotinylation-tag, or an antibody binding module. We demonstrated the functional assembly of viruses with all three modifications.

In addition, we established two N-terminal fusions to the capsid proteins using the binding scaffolds of DARPins and Affibodies. To our knowledge, neither the use of these motifs in a viral setting nor the tumor targeting of rAAV by N-terminal fusion have been demonstrated so far.

We applied a wide range of techniques to achieve our goals. Cloning was performed in E. coli, viruses were produced in a human kidney cell line, and viral infections were tested in further human cell lines. Viruses were purified by liquid chromatography and virus samples were analyzed by quantitative real-time PCR and ELISA. Expression of virally delivered reporter genes was analyzed by fluorescence microscopy including time-lapse imaging. Expression and cell viability upon infection was also assessed by flow cytometry and MTT-assays. Viral samples were additionally analyzed by atomic force microscopy and electron microscopy.

Last but not least, we provide an extensive lab journal, a well documented set of over 100 BioBricks and a detailed manual for the Virus Construction Kit.


Our key achievements are:
  • A BioBrick set and assembled plasmids for gene delivery.
    This set comprises the AAV ITRs, a CMV and a hTERT promoter, as well as the genes of interest mVenus, mCherry as reporters and thymidine kinase and cytosin deaminase constructs for prodrug activation, as well as a beta-globin intron and a hGH polyadenylation tag to control gene expression.
  • A Biobrick set and plasmids for viral capsid production and modification.
    This set comprises an Affibody and a DARPin targeting the EGF-receptor and a set of linkers which can be utilized for N-terminal fusions. A modified gene coding for viral capsid proteins, which is modified for the fusion. In addition we provide a viral capsid gene for loop modification and the fitting loop modifications coding for a His-tag, biotinylation sequence, and antibody binding site.
  • An extensive set of experiments demonstrating the functionality of our constructs.



Highlights

Modularization

We converted a recombinant Adeno-associated virus to the BioBrick standard. This modularization is grouped into two parts: first, the generation of a gene of interest (vector) plasmid and second, the generation of vectors for the modified capsid proteins.

Gene of Interest (GOI) Plasmid
The Virus Construction Kit enables researchers to encapsidate virtually any given DNA sequence into AAV-2 particles. As one example from therapeutic focus of our project, prodrug-activating enzymes are provided within the kit for efficient tumor cell killing. Additionally included fluorescent proteins allow monitoring of transduced cells by fluorescence microscopy and flow cytometry. Transgene expression can be fine-tuned using promoters of different specificity and enhancer elements also provided. more

Capsid Plasmids
AAV-2 genes essential for the production of viral particles in a specialized cell line were identified by literature search, isolated on genetic level and modified to meet the requirements of BioBrick assembly. For this purpose, a specialized variant of the iGEM default backbone was created, proven functional and submitted. more

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Targeting

Retargeting of the viral particles was achieved by knocking down the natural tropism of the Adeno-associates virus particles and at the same time introducing capsid modifications for specifically targeting tumor cells. For this purpose, two different strategies were developed, which were targeting via loops or targeting via fusion to the N-terminus of the viral protein VP2.

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Arming

The specifically targeted tumor cells were killed by prodrug activation approaches. Viral particles were charged with thymidine kinase or cytosine deaminase constructs to kill cancer cells upon delivery of ganciclovir or 5-Fluorocytosine, respectively. [more]

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Tumor Killing

Finally we successfully combined different modifactions of the therapeutic viral vector for optimizing it for tumore therapy application. These modifications include the knock down of the natural tropism for HSPG receptor, the tumor targeting using the affibody ZEGFR1907 fused to the N-terminus of the viral coat protein VP2 and an encapsidated vectorgenome that was bricked down and reassembled containing the prodrug converting Guanosine Monophosphate Kinase Thymidine Kinase fusion protein (mGMK-TK).

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Modeling

Efforts in the lab were accompanied by mathematical modeling of the viral production and infection and the attempt to fit the models to time-lapse microscopy data. Planning of fusion proteins and loop modifications was based on structural analysis and modeling of the viral capsid, based on known crystal structrues. [more]

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