By: Emmette Hutchinson, PhD
Synthetic biology is an interdisciplinary field that utilizes an engineering approach to construct novel biological products, circuits and designer organisms. This field has the potential to revolutionize many aspects of society from chemical production to healthcare. Synthetic biology holds particular promise in the production of biological therapeutics or chemical compounds for the treatment of disease. Increased efficiency and stability of production can be especially beneficial when treating global diseases that are typically associated with poverty. Treatment for these conditions is typically funded by grants from large charitable foundations, sometimes leading to scarcity as funding recedes.
In 2015, 212 million cases of malaria were reported worldwide, predominantly among the poorest countries in the world. While major initiatives such as the President’s Malaria initiative and the Gates foundation focus on various aspects of combating the disease, such as the spread of the parasite and the eradication of the disease, respectively, cost-effective treatments for infections are still needed. The most efficacious treatments for malaria are artemisinin-based combination therapies (ACTs). The 2015 Nobel Prize in Medicine was awarded in part to Youyou Tu for her work demonstrating that artemisinin, an Artemisia annua (sweet wormwood) extract, was an effective anti-malarial treatment. Landmark research published in 2006 demonstrated synthetic production of artemisinic acid, a precursor to artemisin, in yeast. Prior to this study, the only source of artemisinin was tiny hairs found on the surface of the wormwood. The supply of artemisinin has previously been unstable, resulting in dramatic price fluctuations. These price spikes have resulted in both shortages and unattainable cost of treatment. The pharmaceutical giant Sanofi licensed the yeast strain with the hope of creating a more reliable source of artemisinin. In part, due to market forces pushing down the price of artemisinin (primarily a surge in world-wide Artemisia annua cultivation), Sanofi recently sold both its technology and production facilities to Huvepharma. Despite the potential of synthetic biology to disrupt the pharmaceutical industry, this is an example of how existing production methods can impede adoption of more efficient (and stable) synthetic approaches. An alternative to synthetic production of artemisinin in yeast, termed COSTREL (combinatorial supertransformation of transplastomic recipient lines), re-creates the enzymatic pathway necessary to produce artemisinin in tobacco. Although not as efficient as synthetic production of the chemical in yeast, this route offers a significant per-acre boost in artemisinin production over the native source and a potentially more open market to supply drug manufacturers.
Similar to malaria, snake bites predominantly affect impoverished regions of the world. This makes the use of life-saving anti-venoms particularly burdensome as they are both expensive and difficult to produce. The World Health Organization estimates that up to 2.5 million cases of envenoming occur each year, resulting in death, amputations and permanent disabilities. Antivenoms are typically produced using plasma from hyperimmune animals, an often expensive and time-consuming process. In some cases, the profit margins are considered too low to continue producing effective antivenoms such as FAV-Afrique, a polyvalent antivenom effective against 10 species of sub-Saharan snakes. Two recent approaches utilizing synthetic antibody fragments have shown promising effects for protection against specific snake venoms. In a screen for antibody fragments to snake venom, Prado and colleagues found two fragments that protected mice against muscle damage from Bothrops jararacussu and Bothrops brazili venom. Ramos and colleagues designed two synthetic DNA sequences encoding components of coral snake (Micrurus corallinus) venom. Serum obtained from animals immunized with these DNA sequences resulted in 60% survival of animals given a lethal dose of coral snake venom. This second approach eliminates the need for difficult-to-obtain venom when seeking to generate hyperimmune animals as anti-venom producers. It is possible that these or similar synthetic biology approaches could be utilized to produce FAV-Afrique or other polyvalent antivenoms in a faster, more cost-effective manner than hyperimmune animals.
While the possibility of artemisinic acid-producing yeast, high artemisinin-yielding tobacco, and more efficient sources of anti-venom are compelling, regulatory challenges and ethical dilemmas are abundant in the burgeoning field of synthetic biology. Both the US and the EU have recently held surveys and drafted opinions concerning the ethics and risks of synthetic biology. One potential issue with the use of synthetic biology approaches to industrial scale production of chemicals or recombinant proteins is the potential for uncontained spread of the recombinant organism or uncontrolled transfer of the modified genetic material. Another concern centers around the impact of synthetic biology on existing biological diversity. There are also concerns regarding the proliferation of synthetic biology capabilities and biosecurity. At the moment, the United States is in middle of an epidemic of opioid addiction. Synthesis of more complex chemicals in yeast also opens up issues with substance control. A research group has already demonstrated the ability to synthesize heroin in yeast, cheaply and effectively in much the same manner as one might brew beer, raising the possibility that new, designer substances of abuse could be produced in a similar manner. Approaches to the issue of biocontainment have varied, but as the control of synthetic transcriptional circuits becomes robust, more efficacious approaches to biocontainment can be developed. One recent approach to this problem implemented a two-part genetic version of a Dead Man’s Switch into E. coli, which will kill the synthetic organism when certain conditions are not met. As a standard operating procedure, this system would go a long way toward addressing containment of engineered organisms.
The engineering of novel biologicals, re-purposing of existing or development of new transcriptional circuits and entirely new organisms holds immense promise for all aspects of society. These technologies will likely impact the treatment of diseases typically associated with poverty initially, as the increased efficiency of production will lead to stability in price and decreased scarcity of therapeutics. Once concerns of containment and potential effects on existing ecosystems are sufficiently addressed, the broad application of these technologies becomes more reasonable. As the methodologies enabling the creation of designer organisms and novel biologicals improves, the market forces that impede adoption of more efficient synthetic sources of therapeutics may also have less of an impact.
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By: Sarah L Hawes, PhD
Diseases transmitted through contact with an animal carrier, or “vector,” cause over one million deaths annually, many of these in children under the age of five. More numerous, non-fatal cases incur a variety of symptoms ranging from fevers to lesions to lasting organ damage. Vector-borne disease is most commonly contracted from the bite of an infected arthropod, such as a tick or mosquito. Mosquito-borne Zika made recent, regular headlines following a 2015-2016 surge in birth defects among infants born to women bitten during pregnancy. Other big names in vector-borne disease include Malaria, Dengue, Chagas disease, Leishmaniasis, Rocky Mountain spotted fever and Lyme.
Vaccines do not exist for many of these diseases, and the Centers for Disease Control (CDC) Division of Vector-Borne Diseases focuses on “prevention and control strategies that can reach the targeted disease or vector at multiple levels while being mindful of cost-effective delivery that is acceptable to the public, and cognizant of the world’s ecology.” Prevention through reducing human contact with vectors is classically achieved through a combination of physical barriers (i.e. bed nets and clothing), controlling vector habitat near humans (i.e. dumping standing water or mowing tall grass), and reducing vector populations with poisons. For instance, the Presidential Malaria Initiative (PMI), initiated under President Bush in 2005, and expanded under President Obama, reduces vector contact through a complement of educating the public, distributing and encouraging the use of bed nets, and spraying insecticide. Now a 600 million dollar a year program, PMI has been instrumental in preventing several million Malaria-related deaths in the last decade.
But what if a potentially safer, cheaper and more effective solution to reduce human-vector contact exists in the release of Genetically Modified (GM) vector species? Imagine a mosquito engineered to include a new or altered gene to confer disease resistance, sterility, or to otherwise impede disease transmission to humans. Release of GM mosquitos could drastically reduce the need for pesticides, which may be harmful to humans, toxic to off-target species, and have led to pesticide-resistance in heavily-sprayed areas. Health and efficacy aside, it is impossible to overturn or poison every leaf cupping rainwater where mosquitos breed. GM mosquitos could reach and “treat” the same pockets of water as their non-GM counterparts. However, an insect designed to pass on disease resistance to future generations would mean persistence of genetic modifications in the wild, which is worrisome given the possibility of unintended direct effects or further mutation. An elegant alternative is the release of GM vector animals producing non-viable offspring – and this is exactly what biotech company Oxitec has done with mosquitos.
Oxitec’s OX513A mosquitos express a gene that interferes with critical cellular functions in the mosquitos, but this gene is suppressed in captivity by administering the antibiotic tetracycline in the mosquitos’ diet. Release of thousands of non-biting OX513A males into the wild results in a local generation of larvae which, in the absence of tetracycline, die before reaching adulthood. Release of OX513A has proven successful at controlling mosquito populations in several countries since 2009, rapidly reducing local numbers by roughly 90%. Oxitec’s OX513A line may indeed be a safe and effective tool. But who is charged with making this call for OX513A and, moreover for future variations in GM vector release?
Policy governing use of genetically modified organisms must keep pace with globally available biotechnology. Regulatory procedures for the use of GM vector release are determined by country, and there is a high degree of international policy alignment. The Cartagena Protocol on Biosafety is a treaty involving 170 nations currently (the US not included) that governs transport of “living modified organisms resulting from modern biotechnology” with potential to impact environmental or human health. The World Health Organization (WHO) and the Foundation for the National Institutes of Health (FNIH) published the 2014 guidelines for evaluating safety and efficacy of GM mosquitos.
Within the US, the 2017 Update to the Coordinated Framework for the Regulation of Biotechnology was published this January in response to a solicitation by the Executive Office of the President for a cohesive report from the Food and Drug Administration (FDA), Environmental Protection Agency (EPA), and US Department of Agriculture (USDA). Separately, biotech industry has been given fresh guidance on whether to seek FDA or EPA approval (in brief): if your GM product is designed to reduce disease load or spread, including vector population reduction, it requires New Animal Drug approval by FDA; if it is designed to reduce pest population but is un-related to disease, it requires Pesticide Product approval by EPA under the Federal Insecticide, Fungicide, and Rodenticide Act.
Thus, for a biotech company to release GM mosquitos in the US with the intent of curbing the spread of mosquito-borne disease, they must first gain FDA approval. Oxitec gained federal approval to release OX513A in a Florida suburb in August 2015 because of FDA’s “final environmental assessment (EA) and finding of no significant impact (FONSI).” These FDA assessments determined that the Florida ecosystem would not be harmed by eliminating the targeted, invasive Aedes aegypti mosquito. In addition, they affirmed that no method exists for the modified gene carried by OX513A to impact humans or other species. Risks were determined to be negligible, and include the accidental release of a few, disease-free OX513A females. For a human bitten by a rare GM female, there is zero risk of transgene transfer. There is no difference in saliva allergens, and therefore the response to a bite, from GM and non-GM mosquitos. In addition, as many as 3% of OX513A offspring manage to survive to adulthood, presumably by spawning in tetracycline-treated water for livestock. These few surviving offspring will not become a long-term problem because their survival is not a heritable loop-hole; it is instead analogous to a lucky few mosquitos avoiding contact with poison.
Solid scientific understanding of the nature of genetic modifications is key to the creation of good policy surrounding the creation and use of GMOs. In an updated draft of Guidelines For Industry 187 (GFI 187), the FDA advises industry seeking New Animal Drug Approval to include a molecular description of the intentional genetic alteration in animals, method for alteration, description of introduction to the animal, and whether the alteration is stable over time/across generations if heritable, and environmental and food safety assessments. Newer genomic DNA editing techniques such as CRISPR offer improved control over the location, and thus, the effect of genetic revisions. In light of this, the FDA is soliciting feedback from the public on the GFI 187 draft until April 19th, 2017, in part to determine whether certain types of genetic alteration in animals might represent no risk to humans or animals, and thus merit reduced federal regulation.
Following federal clearance, the decision on whether to release GM vectors rests with local government. Currently, lack of agreement among Florida voters has delayed the release of OX315A mosquitos. Similar to when GM mosquito release was first proposed in Florida following a 2009-2010 Dengue outbreak, voter concern today hinges on the perception that GM technology is “unproven and unnatural.” This illustrates both a healthy sense of skepticism in our voters, and the critical need to improve scientific education and outreach in stride with biotechnology and policy. Until we achieve better public understanding of GM organisms, including how they are created, controlled, and vetted, we may miss out on real opportunities to safely and rapidly advance public health.
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