Difference between revisions of "Template:Freiburg/PageTest5"

 
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<link href="https://fonts.googleapis.com/css?family=Josefin+Sans:100" rel="stylesheet">
 
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             <h1 align="center">CAR T Cells</h1>
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             <h1 align="center">Human Practice</h1>
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            <div class="item"><p>
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            CAR T cells are engineered <a href="https://2017.igem.org/wiki/index.php?title=Template:Freiburg/PageTest3#666">T cells</a> that are expressing a specific chimeric antigen receptor (CAR) and have demonstrated great potential in fighting cancer. CAR is composed of a recognition domain derived from monoclonal antibodies and a signaling domain including CD3- and costimulatory domains. The recognition domain of the CAR targets specific molecules expressed in cancer cells and therefore triggers remarkable responses including activation and killing activity of T cells mediated by the signaling domain. As one approach of adoptive cell transfer, CAR T cell therapy shows reduced toxicity because a patient's own immune cells are collected and modified to treat their cancer.
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            <p>
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Public outreach efforts are necessary for science in order to enhance the general understanding of science and to facilitate acceptance. In return, the scientific community can greatly profit from the bidirectional dialogue. <br>
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The first possibility to present our project to the commmunity was the science fair in the heart of Freiburg, where local institutes could present their research focus to an interested audience from young to old. People showed a lot of interest towards our project, recognizing the importance and the potential of Synthetic Biology in health care as well as therapeutics and were happy to have a dialogue with us.  
 +
                </p>
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                <p>
 +
While being impressed by our project design, many people showed misinformation and uncertainties towards the basics of the immune system. We faced a lot of questions concerning genetic modification as well as immunobiology and vaccination in general.
 +
Inspired by these conversations we conducted a survey, with the purpose to find out where such uncertainties might originate from. By offering the chance to win attractive prizes (thanks to our sponsors!), we could gather a lot of different opinions on controversial topics like vaccination and genetic modification.<br>
 +
We presented the results of the study along with our further elaborated project on the Science Days. This is an annual science fair especially oriented for a younger audience like school classes, hosted by the Science Days Club and the Europapark in Rust, one of the biggest amusement parks in Europe. Based on the experience of the science fair, we again engaged the people in a dialogue concerning our project and the basics of immunology.
 +
                </p>
 +
                <p>
 +
In summary, we grasped the chance to inform the public about Synthetic Biology and especially the chances it offers, concretely about CARTELTM. In return, we received valuable input to modify and influence our work by improving the safety aspect, which showed that Synthetic Biology can greatly profit being accessible to the public. To see more about that, see our Integrated Human Practices section
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                </p>
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            </div>
 
              
 
              
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            <p>How does CAR T cell therapy work?</p>
 
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                <img src="https://static.igem.org/mediawiki/2017/9/91/T%E2%80%93FREIBURG%E2%80%93CAR_T_cells_activated_in_TM.png" alt="CAR_T_cells-therapy_scheme" height="" width="">
 
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                <div class="figurecaption col-sm-12">   
 
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                    <p><strong>Figure 1: T cells are first collected from a patient via apheresis, then cultured and engineered in the laboratory. These engineered T cells express constitutively CAR and are then infused into the patient. </strong></p>
 
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          <p> In the last few years, CAR T cell therapy has achieved much progress. One CAR T cell therapy from Novartis was approved by the Food and Drug Administration in August 2017 for children and young adults with acute lymphoblastic leukemia (ALL). However, it is still under investigation precisely how CAR T cells work and the potential side effects need to be avoided. We thereby hope to tackle into this developing field of synthetic immunotherapy, helping to make this promising approach safer. So far most CARs are primarily used against hematopoietic cancers and not solid cancers. Our project is aiming at controlling the expression of CAR in certain tumor microenvironment, which may allow the development of CARs against solid tumors.
 
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                <button class="accordion">Science fair</button>
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                        <br>
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                        <div class="item">
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                            <p>                   
 +
                            Our Team was given the chance to participate at the science fair 2017, an biennial event in the heart of the city to promote local institutes and science projects.
 +
                            </p>
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                        <div class="item">
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                            <h3>Synthetic Biology and iGEM</h3>
 +
                            <p>
 +
                            We took the opportunity to represent iGEM and Synthetic Biology in general and to explain our project to curious visitors. There was a huge interest in our project and people liked the idea, because we informed them about the need of a therapy which is effective and has a low risk for serious side-effects and long-term damage.
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                            </p>
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                            <h3>DNA extraction from fruits</h3>
 +
                            <p>
 +
                            For young scientists we offered a DNA extraction from fruits by materials commonly found in households and taught them about the properties of the DNA and its building blocks, the nucleotides. Not only the kids, but also their parents were impressed since they don't get the opportunity to see DNA with the naked eye everyday.
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                            </p>
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                        <div class="item"> 
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                            <h3>Puzzle of Immunology</h3>
 +
                                <p>We also spread awareness for the functionality of the immune system with a puzzle termed ‘Immunologie für Jedermann’ (immunology for everybody) which was gratefully provided by the German Society for Immunologie (Deutsche Gesellschaft für Immunologie e.V, abbr. DGFI). We talked to people about the importance of vaccinations and faced their questions and insecurities by explaining, how the immune system actually works. We had great success which was visible by the huge interest the people showed towards our booth, our project and synthetic biology in general. A lot of them promised to keep track of our project and wanted us to keep them informed.
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                            </p>
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                <button class="accordion">Survey</button>
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                 <h1 align="center">Tumor Microenvironment</h1>
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                      <br>
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                        <div class="item">
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                          <p>
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                            Based on the experience from the science fair, where we received huge recognition for our project, we faced a lot of uncertainty and misbelief concerning the functions of the immune system, vaccination and Synthetic Biology. For that reason we decided to conduct a survey to find out, where such uncertainty might originate from and how to face them. We got feedback from over 140 participants from different countries, who willingly shared their beliefs and opinions.
 +
                            </p>
 +
                          </div>
 +
                    </div>
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                 <button class="accordion">Science days</button>
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                    <div class="panel panel-default panellarge">
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                        <br>
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                        <div class="col-md-4 text align">
 +
                        <img src="http://www.science-days.de/aktuell/Logo_ScienceDays%20ab%202016.jpg" style="text-align: center; width: 300px">
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                        </div>
  
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                <br>
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                            <p>
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                <p>Tumor microenvironment (TM) refers to the cellular environment surrounding a tumor, which includes blood vessels, normal cells and molecules. The interaction between a tumor and its microenvironment results in different physiological processes providing both beneficial and adverse consequences for tumorigenesis <span class="italic">(Quail & Joyce, 2013)</span>. Gene expression is altered in tumor cells to secrete molecules such as cytokines and growth factors and cellular components for instance exosomes to recruit stroma and vascular cells <span class="italic">(Quail & Joyce, 2013; Mittal et al., 2014)</span>. Regardless of different tumor types and their tissue of origin there are some general hallmarks that govern tumorigenesis. Various immune effector cells can also be found in tumor microenvironment, yet their anti-tumor functions are downregulated due to tumor-derived signals such as cytokines <span class="italic">(TL Whiteside, 2013)</span>. Hypoxia is defined as the reduction or lack of oxygen in organs, tissues or cells <span class="italic">(Wu D. & Yotnda P., 2010)</span>. Hypoxia is a constantly evolving participant in overall tumor growth <span class="italic">(Patel & Sant, 2016; Kim Y et al.,2009)</span> and remains the predominant regulator of angiogenesis in the tumor microenvironment <span class="italic">(Mittal et al., 2014)</span>. Tumor angiogenesis is normally induced by a tumor to generate its dedicated blood supply for oxygen and other nutrients by secretion of various growth factors including vascular endothelial growth factor (VEGF). VEGF promotes growth of blood vessels of a tumor and is regarded as constituent element of the tumor microenvironment <span class="italic">(Mittal et al. 2014)</span>. In addition, acidic extracellular pH is a major feature of tumor tissue due to lactate secretion from anaerobic glycolysis and CO<sub>2</sub> from the pentose phosphate pathway <span class="italic">(Kato et al. 2013)</span>. Those factors that determine the nature of tumor are considered to develop targeted therapies. In our project, three of the most common characteristics of tumor microenvironment are chosen to implement one AND-Gate and control the expression of CAR.
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                <h2>Hypoxia</h2>
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                <p>The oxygen supply provided by blood vessels is designed to support the regular amount of cells in the tissue. When a tumor is present in the tissue, the oxygen level is not sufficient to supply the cells in and around the tumor with enough oxygen. This oxygen depletion occurs due to the high metabolism and proliferation rate of tumor cells. The result is hypoxia in the microenvironment of tumors. It was shown that HIF1α, a transcription factor of HRE, gets stabilized under hypoxia.
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                          <div class="item">
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                <h2>pH</h2>
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                <p>As a result of the hypoxic conditions in the tissue around the tumor, the cells switch to anaerobic metabolism, which leads to the secretion of lactic acid. The effect is a lowering of the pH value. In our project we make use of the ability of cells to sense low pH. A low pH activates the TDAG8 receptor, thus triggering a signaling cascade which ends in the activation of pCRE.
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                <h2>VEGF (Vascular Endothelial Growth Factor)</h2>
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                <p>VEGF is often found in high concentrations in the microenvironment of tumors. Tumor cells secrete VEGF in order to trigger the formation of new blood vessels thereby overcoming hypoxia. High VEGF concentrations are detected by the VEGFR2-receptor leading to a signaling cascade which forms the transcription factor NFAT. pCTLA4 is then activated via NFAT.
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<h1 align="center">AND Gate</h1>
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            <br>
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            <p>
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            AND gates are part of logic circuits which are often used in synthetic biology (Wang et al., 2015); the idea is to integrate inputs into outputs. In case of the AND gate two inputs will be integrated to generate one output.
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            AND gates are often used in modeling in order to establish the necessary or optimal amount to create the output. For this the following has to be true: if two inputs are given (+/+), the output is ‘ON’. In case only one input (+/- or -/+) or none (-/-) is given, the output is ‘OFF’. This system can be put into logic functions (Lomnitz, J. G. and Savageau, M. A., 2016).
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            </p>
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            <p>
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            In our project we used this approach for a controlled expression of our output, the chimeric antigen receptor (CAR). The two inputs used to generate this output are high VEGF concentrations and hypoxic conditions or alternatively low pH and hypoxic conditions.
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            In case that only one of the two inputs is given, the CAR will not be expressed and the CAR T cell won’t be able to bind to its target antigen.
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            Through the dependent expression of the CAR, a higher specifity to tumor cells should be achived considering that both VEGF and hypoxic conditions or a low pH and hypoxia should only be given in the direct tumor microenvironment. This will lead to an improvement of the safety of immunotherapy.
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            </p>
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            <p>
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            #### Grafik bitte von einem AND-gate + Projekt (Ada)<br />
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            ### Link zu TM und CAR <br />
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                        <h2>Hypoxia</h2>
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            <p>
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            Due to lowered oxygen concentrations in the tumor microenvironment the cells have to respond to hypoxia (1% oxygen) and gene expression has to be activated. For this the cells possess HIFs (hypoxia-inducible factors), which are heterodimeric transcription factors and are composed of a hypoxia dependent α subunit and a constitutively expressed β subunit (Pescador, N. et al., 2005). <br />
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            In our project we focused on HIF1, however there are also HIF2 and HIF3. Although they are regulated in a similar way, the function and distribution in the tissue varies (Pescador, N. et al., 2005). <br />
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            Under normoxic conditions HIF1α is hydroxylated and is marked by the E3 ubiquitin ligase which leads to the degradation by the proteasome. However, in hypoxic conditions HIF1α is stabilized and can heterodimerize with HIF1β. HIF1 can then bind to hypoxia response elements (HRE) in the nucleus and lead to the expression of the gene of interest (Ziello, J. et al., 2007), which in case of the CARtelTM AND gate the CAR is .
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            </p>
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            <h2>VEGF (Vascular Endothelial Growth Factor):</h2>
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            <p>
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            VEGF is one of the chosen inputs of the CATtelTM AND gate. Normally it initiates blood coagulation but in our system is it utilized to drive the expression of HIF1α. For this the endogenous pathway of VEGF is used, which functions as follows:<br />
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            VEGF is primarily recognized by its receptor VEGFR-2 (vascular endothelial growth factor receptor 2) which is often expressed on tumor cells (Miettinen, M. et al, 2013). <br />
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            VEGFR-2 is a receptor tyrosine kinase, leading to phosphorylation steps upon binding of its activator VEGF. Furthermore, intracellular calcium concentrations increase and the phosphatase calcineurin, which is dependent on calcium, is activated. Calcineurin is then able to dephosphorylate the transcription factor NFAT (nuclear factor of activated T cells), which is then able to enter the nucleus (Minami, T. et al., 2013) and lead to the expression of the gene of our interest - in our case HIF1α.
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            </p>
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            <h2>pH</h2>
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            <p>
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            A low pH is another possible input for the AND gate and an alternative to the VEGF input. Extracellular protons can be sensed by the receptor TDAG8 (T cell death-associated gene 8), also called GPR65 (G protein-coupled receptor 65) and lead to the activation of a G protein. The G protein activates the adenylyl cyclase, which is able to convert ATP into cyclic AMP (cAMP). cAMP can bind and activate PKA (protein kinase A), resulting in the release of a subunit that is translocated into the nucleus where it can phosphorylate the transcription factor CREB1 (cAMP-responsive element-binding protein 1). CREB1 can bind to a synthetic CRE promoter (Ausländer, D. et al., 2014), leading to the transcription of HIF1α.
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        </div>  
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                                As iGEM slowly went to an end, we presented our final project on the Science Days, which were hosted in the Europapark in Rust, one of the biggest theme parks in Europe. It is a science fair especially established to bring research and science closer to kids and school classes, and appeal to their joy of discovery. But also adolescents, parents and teachers are targeted and informed about institutes, companies, and education possibilities. This was the optimal opportunity to share our final project to large audience.
 +
                            </p>
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 +
                              <p>  <link href="http://www.science-days.de/">http://www.science-days.de/</link> </p>
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        <h1 align="center">Knockout</h1>
 
  
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<div class="col-sm-6">
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        <br>
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<div class="image_box left" style="float:right">
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        <p>The genetic circuit designed to control chimeric antigen receptor (CAR) [link to page] expression in the tumor microenvironment relies on exclusive control of hypoxia-inducible factor 1 alpha (HIF1α) by the introduced system. Various strategies have been employed to obtain HIF1α deficient cells. CRISPR strategies were applied by transient transfection and knockdown was lentivirally transduced (link to method) followed by sorting (method link) of fluorescence positive cells. Insertion of mutations was assessed by PCR or T7E1 assay (link) and the absence of gene product was confirmed by Western Blot (link).
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<div class="figure" style="width:180px">
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                  <a href="https://2017.igem.org/Team:Freiburg/Methods"><img src="https://static.igem.org/mediawiki/2017/9/90/Freiburg_car_2.png" style="width:180px;height:50px;"> 
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                    <div class="figurecaption-car" style="float:right">
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                      <p><strong>Methods</strong></p>
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<div class="image_box right" style="float:left">
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        <h3>Approach 1: CRISPR/Cas9 with HDR
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        <p>The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) allows precise editing of genes. Cas9 forms a complex with guide RNA (gRNA) and introduces DNA double strand breaks at sites complementary to the gRNA. These are repaired by different endogenous repair mechanisms, dependent on the circumstances. One process called homology-directed repair (HDR) can be triggered by co-transformation with a repair template containing regions homologous to the target.
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        A plasmid kit with Cas9 from S. pyogenes was used to eliminate HIF1α by HDR (sponsored by OriGene). This was conducted with two plasmids containing different gRNAs, both targeting the first exon of HIF1α, close to the transcription initiation site (Fig. X). The reporter genes GFP and puromycin resistance were stably integrated via a repair template.
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        <p>The CRISPR/Cas9 system can also be used without repair template. This forces cells to repair the double strand break by non-homologous end joining (NHEJ), an error-prone process. At the cleavage site nucleotides are randomly added and removed during repair resulting in deletions, substitutions and insertions. This stochastically produces a shift of the translational frame in the gene, ablating the gene product’s function.
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        For this strategy gRNAs were designed using the <a href="https://portals.broadinstitute.org/gpp/public/seq/search" target="_blank">Broad Institute’s Genetic Perturbation Platform (GPP)</a> online tool. Off-target effects were predicted using the program CCTOP. The most promising candidates were inserted into Cas9  (S. pyogenes) plasmids either by class-II restriction enzyme cloning of dsDNA oligomers or by site-directed mutagenesis PCR. Two selection markers, GFP (Cas9-GFP provided by Reth Lab at BIOSS, originally Zhang Lab at MIT) and puromycin resistance (lenti-Cas-puro-v2, provided by Cathomen Lab at CTC, originally Zhang Lab at MIT) were tested.
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        <p>Another technique to inhibit gene expression is RNA interference, where translation is inhibited by antisense RNA binding to target mRNA. This prevents association of the translational machinery and therefore protein production. Our approach involved stable lentiviral transduction of such short hairpin RNA (shRNA) into cells to obtain continuous downregulation of HIF1α mRNA, as opposed to an only transiently decreased expression level caused by treatment with the RNA molecule itself. These shRNAs rely on the endogenous processing mechanisms of cellular regulatory RNA. The endo-ribonuclease Dicer cuts off the hairpin of the palindromic transcript and the dsRNA forms together with several proteins the so-called RNA-induced silencing complex (RISC). It unwinds the dsRNA producing active RNA complementary to the target mRNA.
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        Since the eradication of HIF1A gene product in our project serves to allow control of this gene’s expression by a synthetic construct, it is crucial that the designed shRNA sequences are complementary to endogenous mRNA but not that of the introduced gene. Therefore, untranslated regions of this transcript were used as target for shRNA sequence design. Some sequences were obtained from research group Zuber in Vienna, others designed with GPP as for the CRISPR experiment. The lentiviral transfer plasmid into which shRNA sequences were cloned contained cyan fluorescent protein (CFP) and neomycin resistance markers.
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Latest revision as of 16:59, 26 October 2017

Logo
Overview Motivation CAR T Cells Tumor Microenvironment AND Gate Outlook Achievements References
Members Attributions Partners Contact
Main Project Modeling Applied Design Proof of Concept BioBricks Basic Part BioBrick Improvement Interlab Study
Safety Cloning HIF1A Knockdown Cell Culture Lab Book
Human Practice Integrated Human Practice Collaborations

Human Practice

Public outreach efforts are necessary for science in order to enhance the general understanding of science and to facilitate acceptance. In return, the scientific community can greatly profit from the bidirectional dialogue.
The first possibility to present our project to the commmunity was the science fair in the heart of Freiburg, where local institutes could present their research focus to an interested audience from young to old. People showed a lot of interest towards our project, recognizing the importance and the potential of Synthetic Biology in health care as well as therapeutics and were happy to have a dialogue with us.

While being impressed by our project design, many people showed misinformation and uncertainties towards the basics of the immune system. We faced a lot of questions concerning genetic modification as well as immunobiology and vaccination in general. Inspired by these conversations we conducted a survey, with the purpose to find out where such uncertainties might originate from. By offering the chance to win attractive prizes (thanks to our sponsors!), we could gather a lot of different opinions on controversial topics like vaccination and genetic modification.
We presented the results of the study along with our further elaborated project on the Science Days. This is an annual science fair especially oriented for a younger audience like school classes, hosted by the Science Days Club and the Europapark in Rust, one of the biggest amusement parks in Europe. Based on the experience of the science fair, we again engaged the people in a dialogue concerning our project and the basics of immunology.

In summary, we grasped the chance to inform the public about Synthetic Biology and especially the chances it offers, concretely about CARTELTM. In return, we received valuable input to modify and influence our work by improving the safety aspect, which showed that Synthetic Biology can greatly profit being accessible to the public. To see more about that, see our Integrated Human Practices section


Our Team was given the chance to participate at the science fair 2017, an biennial event in the heart of the city to promote local institutes and science projects.

Synthetic Biology and iGEM

We took the opportunity to represent iGEM and Synthetic Biology in general and to explain our project to curious visitors. There was a huge interest in our project and people liked the idea, because we informed them about the need of a therapy which is effective and has a low risk for serious side-effects and long-term damage.

DNA extraction from fruits

For young scientists we offered a DNA extraction from fruits by materials commonly found in households and taught them about the properties of the DNA and its building blocks, the nucleotides. Not only the kids, but also their parents were impressed since they don't get the opportunity to see DNA with the naked eye everyday.

Puzzle of Immunology

We also spread awareness for the functionality of the immune system with a puzzle termed ‘Immunologie für Jedermann’ (immunology for everybody) which was gratefully provided by the German Society for Immunologie (Deutsche Gesellschaft für Immunologie e.V, abbr. DGFI). We talked to people about the importance of vaccinations and faced their questions and insecurities by explaining, how the immune system actually works. We had great success which was visible by the huge interest the people showed towards our booth, our project and synthetic biology in general. A lot of them promised to keep track of our project and wanted us to keep them informed.


Based on the experience from the science fair, where we received huge recognition for our project, we faced a lot of uncertainty and misbelief concerning the functions of the immune system, vaccination and Synthetic Biology. For that reason we decided to conduct a survey to find out, where such uncertainty might originate from and how to face them. We got feedback from over 140 participants from different countries, who willingly shared their beliefs and opinions.


As iGEM slowly went to an end, we presented our final project on the Science Days, which were hosted in the Europapark in Rust, one of the biggest theme parks in Europe. It is a science fair especially established to bring research and science closer to kids and school classes, and appeal to their joy of discovery. But also adolescents, parents and teachers are targeted and informed about institutes, companies, and education possibilities. This was the optimal opportunity to share our final project to large audience.

http://www.science-days.de/

Methods

Integrated HP