Difference between revisions of "Team:CGU Taiwan/Entrepreneurship"

 
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<h1 class="headline">Leijuvant <small></small></h1>
 
 
<ul class="header-subnav">
 
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan">HOME</a></li>
 
<li><a href="https://2016.igem.org/Team:CGU_Taiwan">HOME</a></li>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Parts">PARTS</a></li>
 
<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Parts">PARTS</a></li>
 
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<div class="circle"><img style="position:absolute;z-index:+1;width:90%;height:95%;margin-left:3%;margin-top:2%;" src="https://static.igem.org/mediawiki/2016/8/8a/CGU_Taiwan--logo9.jpg"></div>
 
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<div style="font-size:60px;color:#9999CC;text-decoration:none;margin-top:30%;">
 
Entrepreneurship
 
Entrepreneurship
 
</div>
 
</div>
 
<br><br>
 
<br><br>
  
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<b>(1) Short introduction of our project</b>
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<b><font size="6px">Motivation and Goal</font></b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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<div style="color:black;text-decoration:none;font-size:18px;margin-right:10%;">
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Transgenic Leishmania that can be inactivated by light exposure acts as a safe carrier to deliver specific antigens to the APCs for T cells and humoral response. Based on this concept, we established a new model system to generate antigen-specific inactive Leishmania adjuvant (Leijuvant).
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One reason that some severe diseases do not have efficient vaccine is the lack of T cell pathway activation. Due to this finding, leijuvant purpose to directly activate CD4 T cell pathway, and further induced better antibody production. Leijuvant prevent people from infectious diseases that do not have proper drugs, including tuberculosis, malaria and hepatitis C. With leijuvant, hundreds and thousands of people will be saved. Leijuvant has several advantages as a vaccine adjuvant, providing us with a new perspective of protecting ourselves. </div>
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</div>
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<br><br>
 
<br><br>
  
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<b>(2) What organisms do we use? </b>
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<b><font size="6px">Business model</font></b>
 
<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
 
<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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Leishmania, DC2.4, E.coli(DH5α, BL21), C57BL/6 mice
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<img src="https://static.igem.org/mediawiki/2016/1/16/EN1.jpeg" width=700px height=400px></img>
 
</div>
 
</div>
 
<br><br>
 
<br><br>
  
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<b>(3) Which part do we use? </b>
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<b><font size="6px">Marketing</font></b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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<div style="color:black;text-decoration:none;font-size:18px;margin-right:10%;">
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Full length of HA and OVA: We used full length sequence of hemagglutinin (HA) of H1N1 and Ovalbumin as our testing antigen that is expressed in Leishmania. <br>
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The number of vaccination per person is increasing while the number of vaccine-preventable diseases is also increasing. This situation accelerates virus recombination and evolution, thus, new approach is necessary for vaccine industry. Furthermore, therapeutic vaccines that target specific antigens develop rapidly in oncology and immune diseases, heralding a new vaccine market.<br>
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5’UTR, 3’UTR, 2.3kb intron: the sequence is originally from Leishmania genome. Act as promotor, terminator, and expression modulating part in our device.
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In our own survey, we made a questionnaire for public to figure out the demands of indirectly customers. For integrated marketing, we also interviewed with several experts of both basic research, industries and the hospital system (<a href="https://2016.igem.org/Team:CGU_Taiwan/Human_Practices_Survey#anchor3">read more</a>). In the future, we will collect information from our main customers in a large scale and further analyze the data to solid our business model.<br>
 
</div>
 
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<br><br>
 
<br><br>
  
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<b>(4) Where do we perform the experiments?</b>
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<b><font size="6px">Law and regulation </font></b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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<div style="color:black;text-decoration:none;font-size:18px;margin-right:10%;">
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Leishmania and DC2.4 culturing and experiments will be performed in a BSL-2 lab within the flow hood in an independent room and requires wearing lab gloves. Once the Leishmania is inactivated it can be manipulated on the open bench. DH5α and BL21 are manipulated in the Laminar flow. Normal experiments are performed on open bench with gloves.
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FDA is the main administrative organ managing pharmaceutical products in Taiwan, following the acts of GMP and ISO. The environmental impact, safety, and production issues are also under our concern. Detail information is written in our integrated HP wiki page.</div>
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</div>
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<br><br>
  
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<b>(5) The training of the lab team</b>  
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<b><font size="6px">Progress</font></b>  
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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<div style="color:black;text-decoration:none;font-size:18px;margin-right:10%;"><br>
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We received lecture of the following topics: registration for the use of biological materials, human pathogens, recombinant DNA experiments, potentially infectious materials, animals handling, the use of biological safety cabinets (BSCs) and other laminar flow benches (LFBs), emergency procedures for incidents, biosafety levels signs and labels, etc. We further received laboratory practices training of biosafety level 1 and 2. All the experiments are under Dr. Yu and Dr. Chang’s instructions.
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<img src="https://static.igem.org/mediawiki/2016/4/4d/EN2.jpeg" width=850px height=400px></img><br><br>
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We have proven that Leishmania could activate CD4 T cell pathway inducing efficient humoral immune response. In Further study, we will change the well-established OVA protein into our target protein from hepatitis C virus, plasmodium and tuberculosis. Since Leishmania can also be used as treatment drugs toward cytotoxic pathway, it is possible that generate both therapeutic and prophylactic drugs toward Leishmania system. Leijuvant targets diseases that are under rare medical conditions which correspond to the applicant qualifications of orphan drug. In order to benefit patients suffering from diseases without efficient drugs, FDA fastens the process of orphan drug development clinical trial phase 3, accelerating the procedure of leijuvant launched on the market and reducing the cost. The orphan drug application will be summited after preclinical trial is completed. Patent application will also be submitted before clinical trial start. In our blue plan, we will finish preclinical trial and clinical trial in 7 and 4 years respectively and launch leijuvant in 2029.
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<br>
 
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<br><br>
 
<br><br>
  
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<b>(6) How do we protect us? </b>
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<b><font size="6px">Leijuvant production (mimic real-world conditions)</font></b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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<div style="color:black;text-decoration:none;font-size:18px;margin-right:10%;"><br>
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Follow the requirements from different biosafety level laboratory strictly, including wearing gloves, lab coats, masks and goggles if required.  
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<img src="https://static.igem.org/mediawiki/2016/0/0a/Leijuvant_%E6%B5%81%E7%A8%8B%E5%9C%96.png" width=850px height=150px></img><br><br>
 +
Suppose our target antigen is an unknown virus. First, enter the antigen sequence into the MHC peptide prediction system, McHug, to analysis the MHC binding affinity and other basic features. And get the antigen sequence of your interest. Construct the sequence into shuttle vector to maximize the production of the antigen. Transfect the vectors into Leishmania through electroporation. Through drug selection we can select the successfully transfected Leishmania and mass culture it. Then we will use double-photo inactivation system to completely remove its infectiousness. Repeat the same process and we can design and produce different antigen specific leijuvant.<br>
 
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<br><br>
  
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<b>(7) Safety concern in our project</b>
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<b><font size="6px">Sponsor</font></b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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<div style="color:black;text-decoration:none;font-size:18px;margin-right:10%;">
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Leishmania is a genus of trypanosomes that are responsible for the disease leishmaniasis, a blood-borne disease that is spread by sandflies. Leishmania culturing and experiments will be performed in a BSL-2 lab within the flow hood in an independent room and requires wearing lab gloves. There is almost no chance of infecting people under this experimental condition since there is no direct contact with Leishmania. Also, Leishmania will not be able to survive once it leaves the culture condition. Once it is photo-inactivated it can be manipulated on the open bench.
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We will solicit cooperation with pharmaceutical companies that produce vaccines and adjuvants, such as GSK, Merck, Novartis, Pfizer and Sanofi. Project could be improved and process more smoothly toward discussion that combine both scientific and business perception.  
 
</div>
 
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<br><br>
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<b>(8) How to verify the safety of leishmania after photo-inactivation</b>
 
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
 
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1. How to conduct the photo-inactivation?
 
 
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Photosensitizers (PS) can be delivered endogenously with delta-aminolevulinate (ALA) and exogenously with phthalocyanines (PC).
 
−
Detailed protocol (link)<br>
 
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With loading ALA, we can see the florescence with the excitation of uroporphyrin in the 12-well tissue culture plate during illumination of long-wave UV lamp (365nm)1(Fig. 1A.). There wasn’t any illumination by UV lamp without loading ALA(Fig. 1B.). After illuminating with long-wave  UV ， After long-wave UV illumination, we then put it under the LED red light with the wavelength of 620~630nm for 10 minutes (Fig. 1C.) and the double photo inactivation is complete. <br><br>
 
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<img src="https://static.igem.org/mediawiki/2016/f/f1/LeishSafety1.png" width=550px height=400px style="border:2px black solid;border-radius:8px;"></img>
 
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<br><br>
 
  
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
 
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2. How to confirm the sucess of photo-inactivation?<br>
 
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font size=2>According to (Chang, K. P., et al. 2016.)2we can use the following method to confirm the safety of photo-inactivated Leishmania.</font>
 
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</div>
 
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:120px;">
 
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<b> (I) Microscopy of flagellar motility</b>
 
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</div>
 
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:170px;">
 
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After illuminating by long-wave UV and red light, you can observe the samples under the microscope. The difference between [+drug, +light]; [+drug, -light]; [-drug;-light] groups can be easily seen. With the illumination of the both chemicals, all six wells of one plate show no flagellar motility under 100x magnification. However, the control group, [+drug, -light] and [-drug;-light], have high motility same as the wild-type Leishmania.
 
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</div>
 
  
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:120px;">
 
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<b>(II) Cultivation for growth (2 weeks)</b>
 
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</div>
 
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:170px;">
 
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To further confirm the complete inactivation of Leishmania, we separated 100μl from each sample, [+drug, +light], [+drug, -light] and [-drug;-light], right after illumination. And add them into 24-Well plate containing M199+10%HI-FB medium. Observed and kept tract of it continuously for two weeks. Except the [+drug, +light] group, the other two groups were moving and increasing normally. As for the [+drug, +light] group, there was no motility, or proliferation of the Leishmania from the beginning to the end of the two weeks.
 
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</div>
 
  
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:120px;">
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<b> (III) 2 months after subcutaneously injecting inactivated Leishmania into mice</b>
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<div class="end2" style="margin-top:220%;">
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</div>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:170px;">
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We subcutaneously injected mouse with108 of photo-inactivated Leishmania on both sides of the back around the shoulders, and injected normal saline as the control group. There was no skin ulcers or inflammation and no any other Leishmaniasis symptoms among the 10 mouse that serve as our experimental group(Fig. 2.). <br><br>
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<img src="https://static.igem.org/mediawiki/2016/6/6d/LeishSafety2.png" width=550px height=400px style="border:2px black solid;border-radius:8px;"></img>
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Reference:<br>
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<li><a href="#anchor1"><b style="color:white;font-size:28px;margin-left:7%;">TOP</a></li>
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1. Dutta, S., et al. (2008). "Transgenic Leishmania model for delta-aminolevulinate-inducible monospecific uroporphyria: cytolytic phototoxicity initiated by singlet oxygen-mediated inactivation of proteins and its ablation by endosomal mobilization of cytosolic uroporphyrin." Eukaryot Cell 7(7): 1146-1157.<br>
+
</ul>
−
2. Chang, K. P., et al. (2016). "New "light" for one-world approach toward safe and effective control of animal diseases and insect vectors from leishmaniac perspectives." Parasit Vectors 9(1): 396.
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<b style="color:white;font-size:28px;margin-top:5%;margin-left:4%;text-decoration:none;">SITE MAP</b><br>
 
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<br><br>
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<img style="background-color:#0D014D;margin-left:20%;border-radius:50%;border:6px solid white;padding:5px;width:40%;height:70%;position:absolute;margin-left:2%;" src="https://static.igem.org/mediawiki/2016/0/03/CGU_Taiwan--leishmania3.jpg">
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<b>(9) The product safety concern in the future</b>
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<ul class="list1" class="margin-left:5%;">
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan"><b style="color:white;font-size:18px;margin-left:20%;">HOME</li></a>
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<u>Environmental concern</u><br>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Achievements"><b style="color:white;font-size:18px;margin-left:20%;">ACHIEVEMENTS</li></a>
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Leishmania culture requires a certain osmolarity and combination of nutrients. Therefore, our transgenic Leishmania will not survive as soon as it leaves the medium.<br>
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There are some potential adverse effects underlying every drug and vaccine. More animal test should be carried out and the most efficient dose in human will be selected before clinical trials. In the clinical study, side effect after immunization will be identified and study design will be modified as well. If our product passes the FDA approval, the report of unexpected effect will be always under inspection.<br>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Parts"><b style="color:white;font-size:18px;margin-left:20%;">PARTS</li></a>
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<b>(10) How would your project be used in the real world?</b>
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Our Leijuvant can act to facilitate both humeral and cellular immune response which may contribute to the development of vaccine nowadays. Combined with our prediction software, the platform can predict the peptides on MHC molecules and help to optimize the peptide presentation which may benefit to engineer the sequence for further research and developing a more efficient vaccine.
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Description"><b style="color:white;font-size:18px;margin-left:20%;">PROJECT</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Design"><b style="color:white;font-size:17px;margin-left:10%;">Design</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Background"><b style="color:white;font-size:17px;margin-left:10%;">Background</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Results"><b style="color:white;font-size:17px;margin-left:10%;">Results</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Proof"><b style="color:white;font-size:17px;margin-left:10%;">Proof</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Notebook"><b style="color:white;font-size:17px;margin-left:10%;">Notebook</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Human_Practices"><b style="color:white;font-size:18px;margin-left:20%;">HUMAN PRACTICES</li></a>
 
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Human_Practices_Survey"><b style="color:white;font-size:17px;margin-left:10%;">Survey</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Human_Practices_Education"><b style="color:white;font-size:17px;margin-left:10%;">Education</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Human_Practices_Communication"><b style="color:white;font-size:17px;margin-left:10%;">Communication</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Entrepreneurship"><b style="color:white;font-size:17px;margin-left:10%;">Entrepreneurship</li></a>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Software"><b style="color:white;font-size:18px;margin-left:20%;">MODELING</li></a>
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Latest revision as of 06:30, 2 December 2016


Entrepreneurship


Motivation and Goal
One reason that some severe diseases do not have efficient vaccine is the lack of T cell pathway activation. Due to this finding, leijuvant purpose to directly activate CD4 T cell pathway, and further induced better antibody production. Leijuvant prevent people from infectious diseases that do not have proper drugs, including tuberculosis, malaria and hepatitis C. With leijuvant, hundreds and thousands of people will be saved. Leijuvant has several advantages as a vaccine adjuvant, providing us with a new perspective of protecting ourselves.


Business model


Marketing
The number of vaccination per person is increasing while the number of vaccine-preventable diseases is also increasing. This situation accelerates virus recombination and evolution, thus, new approach is necessary for vaccine industry. Furthermore, therapeutic vaccines that target specific antigens develop rapidly in oncology and immune diseases, heralding a new vaccine market.
In our own survey, we made a questionnaire for public to figure out the demands of indirectly customers. For integrated marketing, we also interviewed with several experts of both basic research, industries and the hospital system (read more). In the future, we will collect information from our main customers in a large scale and further analyze the data to solid our business model.


Law and regulation
FDA is the main administrative organ managing pharmaceutical products in Taiwan, following the acts of GMP and ISO. The environmental impact, safety, and production issues are also under our concern. Detail information is written in our integrated HP wiki page.


Progress



We have proven that Leishmania could activate CD4 T cell pathway inducing efficient humoral immune response. In Further study, we will change the well-established OVA protein into our target protein from hepatitis C virus, plasmodium and tuberculosis. Since Leishmania can also be used as treatment drugs toward cytotoxic pathway, it is possible that generate both therapeutic and prophylactic drugs toward Leishmania system. Leijuvant targets diseases that are under rare medical conditions which correspond to the applicant qualifications of orphan drug. In order to benefit patients suffering from diseases without efficient drugs, FDA fastens the process of orphan drug development clinical trial phase 3, accelerating the procedure of leijuvant launched on the market and reducing the cost. The orphan drug application will be summited after preclinical trial is completed. Patent application will also be submitted before clinical trial start. In our blue plan, we will finish preclinical trial and clinical trial in 7 and 4 years respectively and launch leijuvant in 2029.


Leijuvant production (mimic real-world conditions)



Suppose our target antigen is an unknown virus. First, enter the antigen sequence into the MHC peptide prediction system, McHug, to analysis the MHC binding affinity and other basic features. And get the antigen sequence of your interest. Construct the sequence into shuttle vector to maximize the production of the antigen. Transfect the vectors into Leishmania through electroporation. Through drug selection we can select the successfully transfected Leishmania and mass culture it. Then we will use double-photo inactivation system to completely remove its infectiousness. Repeat the same process and we can design and produce different antigen specific leijuvant.


Sponsor
We will solicit cooperation with pharmaceutical companies that produce vaccines and adjuvants, such as GSK, Merck, Novartis, Pfizer and Sanofi. Project could be improved and process more smoothly toward discussion that combine both scientific and business perception.





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