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<title>Team: Technion 2015</title>
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<br>
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</head>
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<body>
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<!-------------------------------------nav-bar---------------------------------------------->
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<div class =  "col-md-2" >
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<div class="nav_menu">
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<div class="side">
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<ul>
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<a href="https://2015.igem.org/Team:Technion_Israel" style="position:fixed; width: 170px; display:inline-block; text-decoration: none;" class="nav_home_link">
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<img src="https://static.igem.org/mediawiki/2015/c/c1/Team_Technion_Navbar_Home.png" alt="Home" id="nav_home" /><div class="home_text">Home</div></a>
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<div class =  "col-md-8" >
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<div class="row">
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<div class =  "col-md-12" >
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<h3 id="pr"> The Problem </h3>
 +
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<p>
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Autoimmune diseases and allergies are on the rise. Scientists have developed many theories as to why this is happening: The hygiene
 +
hypothesis
 +
<sup id = "cite ref-1 " class ="reference">
 +
<a href="#ref1" original-title>[1] </a>
 +
</sup>,
 +
increasing usage of antibiotics, certain ubiquitous drugs and more. Regardless of the cause, an estimated 73 million people currently suffer from these diseases in the United States alone. With over $150 billion
 +
dollars
 +
<sup id = "cite ref-2 " class ="reference">
 +
<a href="#ref2" original-title>[2] </a>
 +
</sup>
 +
spent annually on health care for autoimmune diseases and allergies, this dire problem is quickly becoming untenable.
 +
</p>
 +
<br>
 +
 +
<h3 id="im"> The Immune System </h3>
 +
<p>
 +
The immune system is a complex network of tissues, cells, and organs working in tandem to protect our bodies from disease. The Hematopoietic Stem Cell (HSC) is a multipotent, self-renewing, progenitor cell. All differentiated immune and blood cells arise from HSCs that exist in the bone marrow.
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</p>
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<br>
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<br>
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<img class="HPA" src="https://static.igem.org/mediawiki/2017/4/4a/T--TECHNION-ISRAEL--HSC-des.png" alt = "" style= "width: 90%; margin:auto;">
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<br>
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<p style="text-align:center;"> <strong>Figure 1: </strong>Hematopoietic stem cell differentiation tree </p>
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<br>
 +
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<br>
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<h3 id="imt"> Immune Tolerance </h3>
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<p>
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Immune tolerance is the lack of an immunological reaction to the presence of specific substances or tissues that have the capacity to induce an immune response.
 +
<br>
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<br>
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Tolerance is maintained by two mechanisms: Central Tolerance and Peripheral Tolerance. Central Tolerance occurs in the primary lymphoid organs, namely the bone marrow cavities and the thymus, where immature B and T cells, respectively, develop. High affinity interaction between immature lymphocytes and antigens/epitopes present in the bone marrow, or thymus, leads to negative selection, and inactivation, of these reactive immune cells (figure 2).
 +
<br>
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<br>
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Peripheral Tolerance occurs in the immune periphery, after B and T cells exit the primary lymphoid organs, and is mediated by complex and varied interactions with antigen and other immune cells.
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</p>
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<br>
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<br>
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</div>
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</div>
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 +
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<div class="row">
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<div class= "col-md-offset-1 col-md-10">
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<img class="HPA" src="https://static.igem.org/mediawiki/2017/8/83/T--TECHNION-ISRAEL--figure2.png" alt = "" style= "width: 100%; margin:auto;">
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<br>
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<p style="text-align:center;"> <strong>Figure 2: </strong>Central Tolerance within the bone marrow
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<br>
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<i>
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Immature immune cells within the bone marrow undergoing selection. The red cell recognizes auto-antigen and as a result undergoes apoptosis. The orange immune cell does not recognize auto-antigen and is allowed to complete maturation
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</i>
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</p>
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</div>
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<br>
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<br>
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<div class="row">
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<div class= "col-md-12">
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<h3 id="de"> How autoimmune diseases and allergies develop </h3>
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<p>
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The development of immune disease begins with loss of tolerance. In autoimmune disease there is a loss of tolerance to “self” (figure 3) whereas in allergic reactions there is loss of tolerance to specific external antigens which are harmless.  This loss of tolerance is referred to as sensitization. The exact mechanism through which tolerance is lost is still being investigated, and there appear to be multiple pathways involved in the onset of disease.
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</p>
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<div class="row">
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<br>
 +
 +
<p style="text-align:center;"> <strong>Figure 3: </strong>Disease causing immune cell escaping into the bloodstream
 +
 +
<br>
 +
<i>
 +
An immature B-cell capable of recognizing auto-antigen somehow manages to mature and escape into the blood stream. This cell will potentially go on to cause the onset of autoimmune disease
 +
</i>
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</p>
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<br>
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</div>
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</div>
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<div class="row">
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<div class= "col-md-12">
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<h3 id="cu"> Current Treatment Options</h3>
 +
<p>Many treatments for both autoimmune disease
 +
<sup id = "cite ref-3 " class ="reference">
 +
<a href="#ref3" original-title>[3] </a>
 +
</sup>
 +
and allergies,
 +
<sup id = "cite ref-4 " class ="reference">
 +
<a href="#ref4" original-title>[4] </a>
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</sup>
 +
such as immunosuppression and desensitization, have been attempted with varying degrees of success, but have largely been ineffective at curbing the prevalence of these conditions.  A single treatment modality for immune disorders has yet to be developed. Whilst allergic reactions and the many autoimmune diseases are quite distinct in their presentation and pathophysiology, they both stem from the improper, or unnecessary, activation of the immune system. This common etiology allows hope that one day a single treatment may be sufficient to cure or prevent both types of disease.
 +
</p>
 +
 +
 +
<br>
 +
<h3 id="so"> Our Solution</h3>
 +
<p>We intend to design a preventative treatment for allergies and autoimmune disease. Through genetic engineering and subsequent transfusion of hematopoietic stem cells we hope to induce widespread and continuous immune tolerance towards specific antigens that are known causes of allergic and autoimmune disease. By utilizing the natural mechanism of Central Tolerance we intend to “educate” the immune system and make it tolerant to specifically targeted antigens. To do this, we are creating a plasmid based platform that will induce hematopoietic stem cells, and their differentiated lineages, to display target antigen on their membrane with the goal of inducing immune tolerance (figure 4). While the specific antigens we are working on represent only a fraction of the existing diseases and allergies, they serve as proof of concept for a system that we hope will be modular, and effective enough to prevent many more autoimmune and allergic diseases.
 +
</p>
 +
<br>
 +
</div>
 +
</div>
 +
 +
 +
<div class="row">
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<div class= "col-md-offset-1 col-md-10">
 +
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<img class="HPA" src="https://static.igem.org/mediawiki/2017/1/16/T--TECHNION-ISRAEL--DE-3.png" alt = "" style= "width: 100%; margin:auto;">
 +
<br>
 +
 +
<p style="text-align:center;"> <strong>Figure 4: </strong>Hematopoietic Stem Cell induced to present antigen on its surface
 +
<br>
 +
<i>
 +
after transfection with the ToloGen plasmid and genomic integration, the HSC expresses the desired epitopes on its membrane.
 +
</i>
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</p>
 +
<br>
 +
<br>
 +
 +
</div>
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</div>
 +
 +
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<div class="row">
 +
<div class= "col-md-12">
 +
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<h3 id="so"> Prevention is the key</h3>
 +
<p>
 +
Immunological memory poses a unique problem for HSC based gene therapy. Induction of tolerance will ideally prevent the maturation of harmful B and T cells, but the mechanisms involved do not directly affect existing plasma, and memory, immune cells. Only extreme and dangerous conditioning regiments
 +
<sup id = "cite ref-5 " class ="reference">
 +
<a href="#ref5" original-title>[5] </a>
 +
</sup>
 +
can eradicate these cells, and this would be unacceptable in a preventative treatment. Thus, patients already sensitized to self-antigen or external allergens will most likely not be cured, and will almost certainly reject any transplanted cells that present such antigens. For this reason we have chosen to focus on prevention, a fundamentally different and novel method for contending with autoimmune and allergic diseases. The ideal candidates for our proposed treatment are newborn babies who have limited immunological memory and development. HSCs can be harvested from cord blood, genetically engineered in a closed environment cell processing system (Link to integrated HUMAN PRACTICE), and transfused back into the child, all within 2-3 days. Finally, after 2-4 weeks, the genetically engineered HSCs will be induced to display the chosen antigens, engendering immune tolerance towards these epitopes.
 +
</p>
 +
 +
<br>
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<br>
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</div>
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</div>
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<div class="row">
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<img class="HPA" src="https://static.igem.org/mediawiki/2017/f/f9/T--TECHNION-ISRAEL--DE-sc.png" alt = "" style= "width: 80%; margin:auto;">
 +
<br>
 +
<p style="text-align:center;"> <strong>Figure 5: </strong>ToloGen Treatment Strategy
 +
<br>
 +
<i>1. Cord blood is harvested from new born babies. 2. CD34+ HSCs are isolated from the cord blood using magnetic beads and centrifugation. 3. The HSCs are electroporated with the ToloGen plasmid. 4. The HSCs are transfused back into the baby.
 +
</i></p>
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<br>
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</div>
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</div>
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<div class="row">
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<div class= "col-md-12">
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 +
<h3 id="vi">Vision</h3>
 +
<p>
 +
In the 20th century it is estimated that smallpox killed over 500,000,000 people. In 1980 the World Health Organization declared smallpox eradicated
 +
<sup id = "cite ref-6 " class ="reference">
 +
<a href="#ref6" original-title>[6] </a>
 +
</sup>. What was once unimaginable had been accomplished, the disease that had killed more people than all human wars put together, was destroyed. This amazing feat was not accomplished with a miracle cure, a previously unknown panacea, but with a vaccine. Sometimes an ounce of prevention, rather than a pound of cure, is the key. Autoimmune and allergic diseases pose an ever-growing medical and fiscal disaster. It is our hope that this research will help lead the way towards a new treatment paradigm for autoimmune and allergic disease. A paradigm founded on prevention.
 +
</p>
 +
<p>
 +
The children of tomorrow will never know the misery wrought upon mankind by the smallpox virus. We dream of a day when they will never know the misery of autoimmune diseases and allergies.
 +
</p>
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<li id="ref1">Okada, H., et al. "The ‘hygiene hypothesis’ for autoimmune and allergic diseases: an update." <i>Clinical & Experimental Immunology  </i>160.1 (2010): 1-9.</li>
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<li id="ref2"> <i> http://www.mdmag.com/medical-news/autoimmune-diseases-cost-us-more-than-100-billion-annually <br> http://www.aafa.org/page/allergy-facts.aspx </i></li>
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<li id="ref3">Alexander, T., et al. "Resetting the immune system with immunoablation and autologous haematopoietic stem cell transplantation in autoimmune diseases." <i> Clin Exp Rheumatol </i>34.4 Suppl 98 (2016): S53</li>
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<li id="ref4">Rosenblum, Michael D. et al. “Treating Human Autoimmunity: Current Practice and Future Prospects.” <i> Science translational medicine </i>4.125 (2012): 125sr1. PMC. Web. 18 June 2017.</li>
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<li id="ref5">Holgate, Stephen T., and Riccardo Polosa. "Treatment strategies for allergy and asthma."<i> Nature Reviews Immunology </i>8.3 (2008): 218-230.</li>
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<li id="ref5">Koplow, David A. Smallpox: the fight to eradicate a global scourge. Univ of California Press, 2004.</li>
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<h1><b>Project Overview</b></h1>
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<div id="big-sentence"><p><b>Our new application of utilizing the human microbiome (genetically modified), is a promising new way to treat male pattern baldness.</b></p></div></br>
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</br><h2>Abstract</h2>
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<p>Hair loss affects roughly 61 million people in the United States.  More than 95% of hair thinning in men is attributed to male pattern baldness, or androgenetic alopecia.  The hair loss product market is a multi-billion dollar market: in the United States alone, this market’s annual revenue is 7 billion dollars, with 3.5% annual growth rate from 2007 to 2012, yet a sufficient solution has yet to be made available. 
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The trigger for androgenetic alopecia is believed to be dihydrotestosterone (DHT), a derivative of testosterone. This summer, we engineered <i>Bacillus subtilis</i>, a bacterium found naturally on the scalp, to secrete 3α-hydroxysteroid dehydrogenase (3α-HSD).  This enzyme reduces DHT to a non-steroidically active compound, using NADPH and NADH as cofactors.</p>
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<p>Our project consists of 3 main parts: 3α-HSD secretion by the <i>Bacillus subtilis</i>, which is a part of the natural microbiome on the scalp, engineered <i>E.coli</i> for NADPH overproduction, and a comb as a tool to create a user-friendly experience. By synchronizing these parts, we hope to be able to show convincing evidence of DHT breakdown, providing a promising platform for treatment of male pattern baldness!</p>
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<h2>Problem</h2>
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<p>Hair loss affects roughly 40 million men and 21 million women in the United States. Approximately 25% of men begin balding by the age 30, and two-thirds begin balding by the age 60. Male pattern baldness (Androgenic alopecia) is the main cause of thinning hair in men - in more than 90% of the cases. This condition is characterized by hair receding from different areas of the scalp, beginning with the lateral sides of the forehead and the vertex<sup><a href="#fn1" id="ref1">1</a></sup>.</p>
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<p>The male sex hormone, testosterone (which is produced largely in the testicles), is partially converted into a more potent derivative: dihydrotestosterone (DHT). The DHT is synthesized from the testosterone in several areas in the body: on the hair follicles, testes and adrenal glands<sup><a href="#fn2" id="ref1">2</a></sup>. The DHT is not only synthesized on the scalp, but is also transported to the scalp through the bloodstream.  It causes the deterioration of the hair follicle through a hormonal cascade<sup><a href="#fn3" id="ref1">3</a></sup>.</p>
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<h2>Available solutions</h2>
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<p>The hair loss product market is a multi-billion dollar market: in the United States alone, this market’s annual revenue is $7 billion, with a 3.5% annual growth rate from 2007 to 2012 <sup><a href="#fn4" id="ref1">4</a></sup>.In China, the world’s second largest economy, sales of minoxidil (one of the two major products on the market), jumped 90% to $16.4 million from 2007 to 2012 <sup><a href="#fn5" id="ref1">5</a></sup>.</p>
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<p>Two major non-surgical treatments are available today:</br>
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<ol><li><b>Finasteride-</b>  Finasteride is one of the leading drugs treating male pattern baldness orally.  It holds 27.5 % of the market in the United States. The pills are taken orally on a daily basis.  The drug blocks the production of DHT, hence reducing its activity on the scalp<sup><a href="#fn1" id="ref1">1</a>,<a href="#fn6" id="ref1">6</a></sup>.  According to a study, after five years of treatment with  Finasteride, 48% of men had increased hair growth, while 75% of men who did not take the drug experienced hair loss<sup><a href="#fn7" id="ref1">7</a></sup>.The main drawback of  Finasteride is it’s potential side effects (partially because the treatment is not a topical one), such as impotence & cancer<sup><a href="#fn8" id="ref1">8</a></sup>.
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<li><b>Minoxidil-</b> holds 28.2 % combined market share in the United States. This is an over-the-counter medication approved for male pattern baldness <sup><a href="#fn1" id="ref1">1</a></sup>. It is rubbed into the scalp twice a day. The mechanism by which minoxidil promotes hair growth is not fully understood. It is most effective for people under 40 years of age whose hair loss is recent. Minoxidil has no effect on receding hairlines. Its use is advised for central (vertex) hair loss only. Hair regrowth can take eight to twelve months. However, only 30–40% of patients experience hair growth<sup><a href="#fn9" id="ref1">9</a></sup>.
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<h2>Our solution strategy</h2>
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<p>With the recent advances that established a strong relationship between the human microbiome and human health and disease, new frontiers in synthetic biology have begun to emerge concomitantly. Namely, rather than engineering a single bacterium chassis as is normally done in conventional synthetic biology applications, we have opted to devise an engineering strategy for a microbiome in order to address health-related problems. </p>
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<p>A national research program of the human microbiome has been established in the United States - The Human Microbiome Project (HMP). The goal of the HMP is to demonstrate that there are opportunities to improve human health through monitoring and/or manipulation of the human microbiome <sup><a href="#fn10" id="ref1">10</a></sup>. New and promising startups have been established based on the idea of the utilization and adaptation of the human microbiome to our needs. For example, AOBiome, LLC. created a dedicated line of products (named ‘Motherdirt’) - the first line of products for the skin made, which includes live bacteria incorporated into the product itself (Ammonia Oxidizing Bacteria) <sup><a href="#fn11" id="ref1">11</a></sup>. This product was publicized extensively in the media - In “The New York Times” and “Good Morning America”. In the past several years, several other big cosmetic companies have released products incorporating natural bacteria - “Clinique Redness Solutions Makeup SPF 15” by Clinique and “Cosmeceuticals Unstress Total Serenity Serums” by Christina are few examples of this promising market <sup><a href="#fn12" id="ref1">12</a></sup>. </p>
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<p>Using this trend in research and cosmetics, we decided to develop a new synthetic biology approach for engineering a microbiome. In our approach, we opt to genetically alter one or more of the bacterial populations that are naturally present to secrete a particular enzyme. Thus, on the one hand we minimally perturb the natural microbiome, while on the other we co-opt it to produce a sustainable treatment of some sort.</p>
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<p>We decided to apply this approach on the problem of male pattern baldness.  We planned to alter the most abundant bacterium which is naturally found on our scalp (<i>Bacillus subtilis</i>) <sup><a href="#fn15" id="ref1">15</a></sup> and genetically engineer it for our needs: taking advantage of its natural protein secretion pathways in order to secrete a DHT-reducing enzyme (as will be explained in the following paragraph). This new application of the human microbiome usage is a path, in our opinion, to solve a variety of conditions and enhance overall wellness in the future.</p>
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<h2>Solution</h2>
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<p>The proposed solution is to incorporate the gene of a DHT-inactivating enzyme into a bacterium that is naturally found on our scalp (<i>B.subtillis</i>). These transgenic microorganisms will be applied on the patient's scalp, and will secrete the enzyme to their environment, providing a constantly renewed supply of the treatment. The proposed DHT-inactivating enzyme is the 3α-hydroxysteroid dehydrogenase (3α-HSD) enzyme. DHT is inactivated by the 3α-HSD to form 3α-androstanediol <sup><a href="#fn13" id="ref1">13</a></sup>, as shown in figure 1 below.</p>
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<figure><img class="img_center" src="https://static.igem.org/mediawiki/2015/e/e1/Technion_Israel_2015_overview-figure1.jpg"/><figcaption>Figure 1:Androgen metabolism in the human prostate<sup><a href="#fn13" id="ref1">13</a></sup></figcaption></figure>
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<p>3α-HSD exists naturally in human prostate, performing the mentioned reaction<sup><a href="#fn14" id="ref1">14</a></sup>.  However, according to previous research, the isoform originated from rat liver has greater specificity to DHT than the human isoform <sup><a href="#fn16" id="ref1">16</a></sup>. Therefore, we chose to use the sequence of <i>AKR1C9</i> for our project.  We altered the sequence in order to delete forbidden restriction sites according to the iGEM regulations for BioBricks.</p>
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<h2>The components of the project</h2>
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<p>An illustration of our proposed solution and project components can be seen in Figure 2.</p>
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<figure><img src="https://static.igem.org/mediawiki/2015/3/3d/Techion_Israel_2015_combined.png" height="550px"/><figcaption>Figure 2: Schematic illustration of the project design, incorporating its main components:  <i>Bacillus subtilis</i> (illustrated by the <span style="color: blue">blue</span> bacteria) which secretes 3α-HSD (illustrated by the <span style="color: gold">yellow</span> circles), engineered <i>E.coli</i> (illustrated by the <span style="color: red">red</span> bacteria) for NADPH molecule (illustrated by the <span style="color: green">green</span> circles) overproduction, and a comb as a tool to create a user-friendly experience. </figcaption></figure>
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<p>In order to better focus on the many different components of our project, we split up into groups to tackle each component: Expression of the 3α-HSD enzyme, secretion of the enzyme, and cofactor production.  Additionally, a group worked on developing a comb- a tool which could provide a user-friendly solution for the consumer in applying our product to the scalp.  For more information about each sub-group, enter the page from the menu above, or visit the links below:</br>
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<ul style="list-style: none"><li><a href="https://2015.igem.org/Team:Technion_Israel/Project/Secretion" target="_blank">Secretion</a></li>
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<li><a href="https://2015.igem.org/Team:Technion_Israel/Project/Secretion" target="_blank">Expression</a></li>
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<li><a href="https://2015.igem.org/Team:Technion_Israel/Project/Cofactor" target="_blank">Cofactor</a></li>
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<li><a href="https://2015.igem.org/Team:Technion_Israel/Design" target="_blank">Comb and Design</a></li>
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<li><a href="https://2015.igem.org/Team:Technion_Israel/Project/Results" target="_blank">Results</a></li></ul></p>
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<h2>Modeling</h2>
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<p>Our novel treatment for male pattern baldness is separated into three modules for modeling analysis, which constitute our entire system:</br>
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<ol><li>The <i>Bacillus</i> – our enzyme producer</li>
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<li>The <i>E.Coli</i> – our cofactor producer.</li>
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<li>3α-HSD – the "engine" of our process.</li></ol></p>
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<p>Since the goal of our system is to break down the DHT molecule, we chose to focus on the enzymatic activity for our model, which has the key role in the breakdown process.</p>
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<p>Using principles from statistical mechanics, we developed a completely new enzyme reaction function and simulated the breakdown process in a way that correlated with our wet lab results.</p>
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<p>Our model allows us to take a look at the mechanism of this enzyme and gain insight into the reactions which eventually break down DHT.</p>
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<p>For more information about our models, click <a href="https://2015.igem.org/Team:Technion_Israel/Modeling" target="_blank">here</a>.</p>
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<h2>What’s next?</h2>
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<p>Our plans in the future consist of examining our product on model mice for androgenic alopecia (male pattern baldness) as a proof of concept. We would like to utilize this model to prove that our combined solution for this condition can actually show results (<i>in-vivo</i>). If this succeeds, we will continue on to perform human trials.</p>
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<p>We strive to open a standalone startup which will take our concepts forward.</p>
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<p>We also believe that our new application of utilizing the human microbiome (genetically modify it for a specific cause ) is a promising new way to treat different conditions.</p></br>
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<sup id="fn1">1. "Hair Loss Statistics." Statistic Brain. Relevant Research, Inc. (International Society of Hair Restoration Surgery), July 27th 2015. (accessed on: July 7th 2015). <http://www.statisticbrain.com/hair-loss-statistics/>.</sup></br>
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<sup id="fn2">2. Takayasu, S., Adachi, K. (1972). The conversion of testosterone to 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone) by human hair follicles. The Journal of Clinical Endocrinology & Metabolism, 34(6), 1098-1101.</sup></br>
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<sup id="fn3">3.  "Changes in Hair Fiber Shape and Size Occur When Pattern Baldness First Develops." Hair Fiber Changes with the Development of Pattern Hair Loss. N.p., 16 Nov. 2002. Web. 07 May 2015. <http://www.androgeneticalopecia.com/hair-loss-biology/hair-loss-fiber-men-women.shtml>.</sup></br>
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<sup id="fn4">4. "Hair Loss Treatment Manufacturing in the US: Market Research Report." Hair Loss Treatment Manufacturing in the US Market Research. OTC Medicines, Sept. 2012. (accessed on: July 7th 2015). <http://www.ibisworld.com/industry/hair-loss-treatment-manufacturing.html>.</sup></br>
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<sup id="fn5">5. Loo, Daryl, and Lisa Pham. "Hair-Loss Drugs' Big Growth in China." Bloomberg Business Week. Bloomberg, June 13th 2013. Web. (accessed on: July 7th 2015). <http://www.businessweek.com/articles/2013-06-13/hair-loss-drugs-big-growth-in-china>.</sup></br>
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<sup id="fn6">6. "Proscar (Finasteride) Drug Information: Description, User Reviews, Drug Side Effects, Interactions - Prescribing Information at RxList." RxList. RxList Inc., n.d. Web. (accessed on: July 7th 2015). <http://www.rxlist.com/proscar-drug.htm>.</sup></br>
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<sup id="fn7">7. "What Is the Success Rate of Propecia? | Hair Loss & Hair Transplant Surgery Guide." Health Development Advice. Health Development Advice - HDA Online, n.d. Web. 07 May 2014. <http://www.hda-online.org.uk/hair-loss/propecia/propecia-success-rate.html>.</sup></br>
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<sup id="fn6">6. "Proscar (Finasteride) Drug Information: Description, User Reviews, Drug Side Effects, Interactions - Prescribing Information at RxList." RxList. RxList Inc., n.d. Web. (accessed on: July 7th 2015). <http://www.rxlist.com/proscar-drug.htm>.</sup></br>
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<sup id="fn8">8. “Propecia Side Effects Center”, RxList <http://www.rxlist.com/propecia-side-effects-drug-center.htm> Last reviewed on RxList June  26th 2015 (accessed on: July 7th 2015).</sup></br>
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<sup id="fn9">9. "Minoxidil Topical: MedlinePlus Drug Information." U.S National Library of Medicine. U.S. National Library of Medicine, 9 Jan. 2010. (accessed on: July 7th 2015). <http://www.nlm.nih.gov/medlineplus/druginfo/meds/a689003.html>.</sup></br>
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<sup id="fn10">10. Peterson, J., Garges, S., Giovanni, M., McInnes, P., Wang, L., Schloss, J. A., ... & NIH HMP Working Group. (2009). The NIH human microbiome project.Genome research, 19(12), 2317-2323.</sup></br>
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<sup id="fn11">11. “What are AOB?”, AOBiome, LLC. 2015 <http://motherdirt.com/aob> (accessed on: July 7th 2015). </sup></br>
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<sup id="fn12">12. “5 Probiotic Skin Cream Choices to Consider”, Beauty Junction Online <http://www.beautyjunctiononline.com/5-probiotic-skin-cream-choices-consider/> Febuary 1st 2105 (accessed on: July 7th 2015).</sup></br>
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<sup id="fn13">13. Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3): The V54L mutation restricting the steroid alternative binding and enhancing the 20α-HSD activity. The Journal of Steroid Biochemistry and Molecular Biology 141, 135-143.
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<sup id="fn14">14. Biswas, M. G.; Russell, D.W.:Expression Cloning and Characterization of Oxidative 17b- and 3a-Hydroxysteroid Dehydrogenases from Rat and Human Prostate. The Journal of Biological Chemistry. 1997, 272, 15959-15966.
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<sup id="fn15">15. Roia, F.C.; Vanderwyk, R., R. W.: Resident Microbial Flora of the Human Scalp and its Relationship to Dandruff. Journal of The Society of Cosmetic Chemists. 1969, 20, 113-134.</sup></br>
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<sup id="fn16">16. Penning, T. M., Jin, Y., Heredia, V. V., & Lewis, M. (2003). Structure–function relationships in 3α-hydroxysteroid dehydrogenases: a comparison of the rat and human isoforms. The Journal of steroid biochemistry and molecular biology, 85(2), 247-255.</sup></br>
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Latest revision as of 21:21, 25 October 2017

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Description



The Problem

Autoimmune diseases and allergies are on the rise. Scientists have developed many theories as to why this is happening: The hygiene hypothesis [1] , increasing usage of antibiotics, certain ubiquitous drugs and more. Regardless of the cause, an estimated 73 million people currently suffer from these diseases in the United States alone. With over $150 billion dollars [2] spent annually on health care for autoimmune diseases and allergies, this dire problem is quickly becoming untenable.


The Immune System

The immune system is a complex network of tissues, cells, and organs working in tandem to protect our bodies from disease. The Hematopoietic Stem Cell (HSC) is a multipotent, self-renewing, progenitor cell. All differentiated immune and blood cells arise from HSCs that exist in the bone marrow.




Figure 1: Hematopoietic stem cell differentiation tree



Immune Tolerance

Immune tolerance is the lack of an immunological reaction to the presence of specific substances or tissues that have the capacity to induce an immune response.

Tolerance is maintained by two mechanisms: Central Tolerance and Peripheral Tolerance. Central Tolerance occurs in the primary lymphoid organs, namely the bone marrow cavities and the thymus, where immature B and T cells, respectively, develop. High affinity interaction between immature lymphocytes and antigens/epitopes present in the bone marrow, or thymus, leads to negative selection, and inactivation, of these reactive immune cells (figure 2).

Peripheral Tolerance occurs in the immune periphery, after B and T cells exit the primary lymphoid organs, and is mediated by complex and varied interactions with antigen and other immune cells.




Figure 2: Central Tolerance within the bone marrow
Immature immune cells within the bone marrow undergoing selection. The red cell recognizes auto-antigen and as a result undergoes apoptosis. The orange immune cell does not recognize auto-antigen and is allowed to complete maturation



How autoimmune diseases and allergies develop

The development of immune disease begins with loss of tolerance. In autoimmune disease there is a loss of tolerance to “self” (figure 3) whereas in allergic reactions there is loss of tolerance to specific external antigens which are harmless. This loss of tolerance is referred to as sensitization. The exact mechanism through which tolerance is lost is still being investigated, and there appear to be multiple pathways involved in the onset of disease.



Figure 3: Disease causing immune cell escaping into the bloodstream
An immature B-cell capable of recognizing auto-antigen somehow manages to mature and escape into the blood stream. This cell will potentially go on to cause the onset of autoimmune disease


Current Treatment Options

Many treatments for both autoimmune disease [3] and allergies, [4] such as immunosuppression and desensitization, have been attempted with varying degrees of success, but have largely been ineffective at curbing the prevalence of these conditions. A single treatment modality for immune disorders has yet to be developed. Whilst allergic reactions and the many autoimmune diseases are quite distinct in their presentation and pathophysiology, they both stem from the improper, or unnecessary, activation of the immune system. This common etiology allows hope that one day a single treatment may be sufficient to cure or prevent both types of disease.


Our Solution

We intend to design a preventative treatment for allergies and autoimmune disease. Through genetic engineering and subsequent transfusion of hematopoietic stem cells we hope to induce widespread and continuous immune tolerance towards specific antigens that are known causes of allergic and autoimmune disease. By utilizing the natural mechanism of Central Tolerance we intend to “educate” the immune system and make it tolerant to specifically targeted antigens. To do this, we are creating a plasmid based platform that will induce hematopoietic stem cells, and their differentiated lineages, to display target antigen on their membrane with the goal of inducing immune tolerance (figure 4). While the specific antigens we are working on represent only a fraction of the existing diseases and allergies, they serve as proof of concept for a system that we hope will be modular, and effective enough to prevent many more autoimmune and allergic diseases.



Figure 4: Hematopoietic Stem Cell induced to present antigen on its surface
after transfection with the ToloGen plasmid and genomic integration, the HSC expresses the desired epitopes on its membrane.



Prevention is the key

Immunological memory poses a unique problem for HSC based gene therapy. Induction of tolerance will ideally prevent the maturation of harmful B and T cells, but the mechanisms involved do not directly affect existing plasma, and memory, immune cells. Only extreme and dangerous conditioning regiments [5] can eradicate these cells, and this would be unacceptable in a preventative treatment. Thus, patients already sensitized to self-antigen or external allergens will most likely not be cured, and will almost certainly reject any transplanted cells that present such antigens. For this reason we have chosen to focus on prevention, a fundamentally different and novel method for contending with autoimmune and allergic diseases. The ideal candidates for our proposed treatment are newborn babies who have limited immunological memory and development. HSCs can be harvested from cord blood, genetically engineered in a closed environment cell processing system (Link to integrated HUMAN PRACTICE), and transfused back into the child, all within 2-3 days. Finally, after 2-4 weeks, the genetically engineered HSCs will be induced to display the chosen antigens, engendering immune tolerance towards these epitopes.




Figure 5: ToloGen Treatment Strategy
1. Cord blood is harvested from new born babies. 2. CD34+ HSCs are isolated from the cord blood using magnetic beads and centrifugation. 3. The HSCs are electroporated with the ToloGen plasmid. 4. The HSCs are transfused back into the baby.


Vision

In the 20th century it is estimated that smallpox killed over 500,000,000 people. In 1980 the World Health Organization declared smallpox eradicated [6] . What was once unimaginable had been accomplished, the disease that had killed more people than all human wars put together, was destroyed. This amazing feat was not accomplished with a miracle cure, a previously unknown panacea, but with a vaccine. Sometimes an ounce of prevention, rather than a pound of cure, is the key. Autoimmune and allergic diseases pose an ever-growing medical and fiscal disaster. It is our hope that this research will help lead the way towards a new treatment paradigm for autoimmune and allergic disease. A paradigm founded on prevention.

The children of tomorrow will never know the misery wrought upon mankind by the smallpox virus. We dream of a day when they will never know the misery of autoimmune diseases and allergies.




  1. Okada, H., et al. "The ‘hygiene hypothesis’ for autoimmune and allergic diseases: an update." Clinical & Experimental Immunology 160.1 (2010): 1-9.
  2. http://www.mdmag.com/medical-news/autoimmune-diseases-cost-us-more-than-100-billion-annually
    http://www.aafa.org/page/allergy-facts.aspx
  3. Alexander, T., et al. "Resetting the immune system with immunoablation and autologous haematopoietic stem cell transplantation in autoimmune diseases." Clin Exp Rheumatol 34.4 Suppl 98 (2016): S53
  4. Rosenblum, Michael D. et al. “Treating Human Autoimmunity: Current Practice and Future Prospects.” Science translational medicine 4.125 (2012): 125sr1. PMC. Web. 18 June 2017.
  5. Holgate, Stephen T., and Riccardo Polosa. "Treatment strategies for allergy and asthma." Nature Reviews Immunology 8.3 (2008): 218-230.
  6. Koplow, David A. Smallpox: the fight to eradicate a global scourge. Univ of California Press, 2004.
My First Website

Department of Biotechnology & Food Engineering
Technion – Israel Institute of Technology
Haifa 32000, Israel

    • igem.technion.2017@gmail.com