Tiffany

Hey everyone! My name is Tiffany Hou and I'm from Hopkinton Massachusetts. I am a rising sophomore at Hopkinton High School. Although I have not taken biology in school yet, I am very interested in the sciences, especially biology because it explains the world around us and is a subject that is always undergoing change. During freshman year, I took a semester of chemistry and a semester of physics which was really nice since it gave me a basic understanding of two subjects in science. As for why I'm here... I heard that this camp has a great reputation and I wanted to get a feel for biology before taking AP bio next year (a little nervous about taking AP bio without a year of bio beforehand). Also, to be honest, I am not really sure about what I want to do in the future and I hope that this camp will either eliminate some career paths or help me realize my true passion.

My hobbies include dancing, listening and playing music, and traveling. I absolutely love to dance and do many styes such as modern, ballet, and Chinese folk dance. Also, in school I am a part of student council, band, the mock trial team, and the math team. Some interesting facts about me (not that interesting.. I'm a pretty normal person) are that I travel to Taiwan every other summer, and I have recently graduated from Chinese school which I have attended for 9 years. I am also very lucky to be a part of a non-profit organization called the Chinese Folk Art Workshop (CFAW), in which we perform around the New England area showcasing Chinese culture. As I mentioned before, I love to dance, and CFAW gives me the wonderful opportunity of both dancing and giving back to society through community service. My community service done through my time in CFAW has not been overlooked; recently, I received the Gold President's Volunteer Service Award.

I am very excited to meet everyone and to start this camp!
 * Research Project: Artemisinin**

__Introduction__ In one year, hundreds of thousands of people die of malaria. It is a major threat to the health of people living in the tropics and subtropics. Many drugs have been developed to fight malaria, but the history of anti-malarial drugs has been marked with the constant struggle between evolving drug-resistant parasites and developing new formulas. One form of treatment, Artemisinin-based Combination Therapies, or ACTs is considered to be the best form of treatment for malaria, but they are largely unaffordable to most of the vulnerable populations. Because of this, scientists are trying to produce Artemisinin through a new process involving synthetic biology, industrial fermentation, and chemical synthesis to lower the cost of Artemisinin and make it available and affordable to the many people who need it.

__Malaria__ About half of the world’s population is at risk for malaria, and most cases and deaths occur in sub-Saharan Africa. However Latin America, Asia, and to a lessened extent the Middle East and Europe are all at risk. About 219 million people are infected with malaria every year, and about 660,000 people die from it.

How Malaria Attacks the Body: The protozoa Plasmodium, a single celled parasite, causes malaria. Malaria enters a human when an infected mosquito bites the person. The parasites, which are called sporozoites at this stage, then pass into the blood. They then circulate in the blood for a few minutes before finding their way into the host’s liver cell, where they divide in the next few weeks or so and produce about 30,000 daughter parasites called merozoites. These merozoites re-enter the bloodstream and enter the protected environment of the red blood cells. When the merozoites are in the host’s red blood cells, they are shielded from detection and attack from the host’s immune system. The merozoites continue to grow and divide, until the red blood cells explode, releasing merozoites into the bloodstream and repeating the whole cycle. The continuous cycle of growth, release, and reinvasion could lead to the destruction of all of the host red blood cells in 12 to 14 days.

__Artemisinin__ Artemisinin is a powerful anti-malarial drug. It is from the plant Artemisia annua, or qinghao that grows in China and Vietnam. It was first developed by Chinese scientists and was first synthesized in 1979. Artemisinin is part of the isoprenoid family, which are organic compounds composed of two or more units of hydrocarbons.

How Artemisinin Works:

Artemisinin works very fast, reducing the parasite’s biomass in the human body, and is good at destroying drug resistant malaria parasites in the bloodstream. It works by attacking the parasite’s sexual stage of its life cycle, which prevents the multiplication of the parasites, which is what fuels the infection. This drug is also the fastest drug that can get rid of the asexual forms of the parasite. The main chemical feature of the drug is the unstable bond between two oxygen atoms, which allows it to produce free radicals that break up proteins in the protozoa. The release occurs upon exposure to Hemozoin, the disposable product formed when the parasite digests blood. The formation of Hemozoin is a necessity in able for the parasite to live. Since Artemisinin disrupts the transfer of the parasite from human to mosquito, it gives hope that Artemisinin will be able to eradicate malaria from areas where it is common.

Problems and Limitations of Artemisinin: Artemisinin is a natural product that has presented a supply chain challenge in the pharmaceutical industry. The plant Artemisinin is extracted from, Artemisia annua, is only planted in China and other regions in Southeast Asia. In addition, it is a very labor- intensive crop that has a lengthy growing cycle of 12-18 months. Artemisia annua also is sensitive to genetic backgrounds, cultivation conditions, and harvesting periods. An estimate of 17,000 ha (hectare 10,000 square meters) is needed to produce enough Artemisinin for 100 million adult treatments. In 2004 there was only an estimate of 4,700 ha of Artemisia grown. Because of this, the demand has outstripped the supply and the shortages have caused the prices to soar. A dose of Artemisinin costs $1.30 whereas another anti-malarial drug, chloroquine, costs $0.25 per dose. Many people needy of an anti-malarial drug, but do not have much money take medicines like chloroquine instead of artemisinin; however, the parasites have become resistant to many of these medicines.
 * Cost

Artemisinins reliably reduce initial malaria parasitaemia, so modeling studies suggest six days of treatment to cure parasite burdens. However, the high rates of recrudescence (10%-15%) seen in artemisinin monotherapy do not match up with the model. These poor results are usually attributed to the short half-life (45 minutes) of artemisinins, which is then further shortened by the increased drug clearance that develops after repeated dosage. Blaming pharmacokinetic factors alone for the poor results of artemisinin monotherapy may not be justified because constant drug levels are not needed for the desired result, and if pharmacokinetic behavior were a problem, prolongation of treatment may be predicted to compensate; however, this has not been observed to be the case.
 * Poor cure rate of monotherapy

Artemisinin has been show to cause brainstem toxicity in animals in pre-clinical experiments; however, millions of doses in various formulations have been given to humans and none of them show signs of major neurotoxicity. It is probably the duration of artemisinin exposure that determines neurotoxicity rather than the maximum concentrations reached. Prolonged high concentrations of artemisinin are not seen in the oral forms, which are the most commonly used forms of artemisinin. There has been a recent claim that artemisinin causes mild but significant hearing loss, which seems to contradict this view. Concern in regard to neurotoxicity should also be maintained in the context of children who have a more vulnerable neurological system and have limited therapeutic experience.
 * Neurotoxicity

The Heroes:

Keasling used synthetic biology to engineer microbes to inexpensively and reliably produce the critical chemical component of artemisinin. Amyris and Sanofi are both companies that are applying their technology to ensure access of affordable malaria treatments worldwide. This has also been funded by a five-year grant awarded by the Bill and Melinda Gates Foundation. Sanofi has also announced that it will ensure its distribution under the “no profit, no loss” principle.
 * Jay Keasling
 * Amyris and Sanofi

WHO is the directing and coordinating authority for health within the United Nations. WHO recommends ACT’s as a malaria treatment and is also looking for ways to make artemisinin more affordable.
 * World Health Organization (WHO)

Derivatives and ACT’s: Artemisinin derivatives are semi-synthetic artemisinin based compounds that improve the effectiveness of artemisinin, since artemisinin has physical properties such as poor bioavailability. Some derivatives include artesunate, which is water-soluble and may therefore be given by injection, artemether, and dihydroartemisinin. ACTs are Artemisinin-based Combination Therapy treatments. They are currently the recommended treatment for malaria. ACT’s are made up of fast acting artemisinin-based compounds combined with a drug from a different class. Companion drugs include lumefantrine, mefloquine, amodiaquine, sulfadoxine/pyrimethamine, piperaquine and chlorproguanil/dapsone, and artemisinin derivatives include artesunate, artemether, and dihydroartemisinin. The benefits of ACTs are their high efficacy, fast action, and the reduced likelihood of resistance developing.

Microbial Production of Artemisinin: By transplanting genes from yeast and from the sweet wormwood tree into //E. coli// bacteria and then by-passing the //E. coli’s// metabolic pathway and engineering a new one based on the mevalonate pathway in yeast, scientists were able to induce the bacteria to produce amorphadiene, a chemical precursor to artemisinin. Scientists are working at elucidating the metabolic pathway in the A. annua plant and identifying the genes needed for artemisinic acid production. They are inserting this pathway into microorganisms and optimizing the resulting microbial strains for commercial production of the precursor through fermentation. The technology needed for this microbial production of artemisinin was developed in the Keasling laboratory. The scientists put bacterial, yeast, and plant genes into a bacterial chassis to create a platform organism capable of synthesizing a lot of isoprenoid precursors. The precursors can then be converted into any isoprenoid product with the addition of a specific biosynthetic gene. In the case of the artemisinin biosynthetic pathway, the gene is amorphadiene synthase. To complete the artemisinin biosynthetic pathway, amorphadiene goes through three oxidation steps to become artemisinic acid. First, Keasling and his group created a new metabolic pathway in the yeast, and then introduced bacterial and A. annua genes into the yeast’s DNA that interacted with the yeast’s own genes to produce amorphadiene. Finally, they cloned the gene from the wormwood tree that produces the enzyme P450, which the plant uses to convert amorphadiene to artemisinic acid, and expressed it in the amorphadiene-producing yeast strain. A very important discovery was the CYP71AV1 gene. This gene accelerates the process of the conversion from amorphadiene to artemisinic acid. Scientists are currently studying how to optimize the CYP71AV1 gene in microbial strains that have been optimized to sustain high-level production of amorphadiene. After this, in the A. annua plant, artemisinic acid is then oxidized to produce artemisinin. However, a singlet oxygen enzyme that can peroxidize artemisinic acid has not yet been discovered, and it is currently postulated that the transformation is preformed in a light-driven reaction. This makes production traditionally seem costly and extremely difficult to perform at a commercial scale. But, at Amyris, an inexpensive chemical process has been developed to overcome those challenges. Using this chemical process, artemisinin can be synthesized exactly replicating its natural material. Once the artemisinin is produced, it must be further chemically converted into derivatives such as artesunate or artemether, which are integrated into ACTs.

How does microbial production of artemisinin solve the problem? Microbial production of artemisinin is much easier than extracting the drug from the plant. Production is also quicker, which lowers the cost of artemisinin significantly, allowing the impoverished populations of Africa and South America who need artemisinin to buy it. Since monotherapy has poor cure rates, and the problem is that some manufacturers are selling artemisinin as a monotherapy instead of a co-therapy as recommended by the WHO, Keasling says that access of the technology to produce artemisinin synthetically can be restricted to responsible manufacturers who will bundle artemisinin as part of the anti-malarial drug “cocktail”. This can also reduce the chances of malaria parasites developing a resistance to artemisinin, since any drug that is used as a monotherapy increases the chances that microbes will develop a resistance to it. Regarding neurotoxicity much is still unknown about the link between artemisinin and neurotoxicity, but taking account of these concerns, artemisinin derivatives have less major toxicity than other available anti-malarial drugs.

Other possible benefits of microbial production of artemisinin: Synthetic artemisinin is anticipated to have an impact on the pervasiveness of counterfeit drugs. Counterfeit drugs have become a major problem and some reports state that about 50% of all drugs on the market in certain parts of Asia and Africa are counterfeited. Providing the market with low cost ACTs could diminish the potential profits made by criminal counterfeiting activities, and the incentive to manufacturer and sell counterfeit anti-malarial drugs could be substantially lowered.