The Mosquito, Synthetic Biology, CRISPR, and Malaria
Using Gene Modification and Gene Drive to Eradicate a Disease
Anthony A. James
Distinguished Professor Dept's of Microbiology & Molecular Genetics and Molecular Biology & Biochemistry, University of California - Irvine
About the Lecture
Population alteration strategies based on transgenic mosquitoes carrying genes that prevent parasite transmission have a role in the malaria eradication agenda. They can consolidate elimination gains by providing barriers to parasite and competent vector reintroduction, and allow resources to be focused on new sites while providing confidence that treated areas will remain malaria-free. A promising strategy for combating malaria is based on harnessing gene drive systems to spread anti-malarial genes throughout mosquito populations rendering them unable to transmit the parasites. Highly-effective anti-malarial gene cassettes that result in a 100% block in mosquitos of Plasmodium falciparum, the most deadly human malaria parasite, were linked to a gene-drive system based on CRISPR/Cas9 biology, and in laboratory experiments using gene drive spread with 99.5% efficiency in Anopheles stephensi mosquitos, the major malarial mosquito vector in urban India. Challenges remain to be overcome before deploying the system against malaria outside the laboratory. Efficacy and safety have to be demonstrated in carefully controlled field trials. Strategic planning in selecting field-sites, engineered mosquito strains, trial designs and implementation strategies will be required to achieve a successful first field trial of this technology. Furthermore, the trials should use local scientists and adhere to stringent community engagement and regulatory standards. And trials should have an epidemiological endpoint that results in local disease elimination.
About the Speaker
Anthony A. James is Distinguished Professor of Microbiology & Molecular Genetic, and Molecular Biology & Biochemistry in the Schools of Medicine and Biological Sciences, respectively, at the University of California, Irvine. Previously, he was a member of the faculty of the Harvard School of Public Health. Anthony works on vector-parasite interactions, mosquito molecular biology, and other problems in insect developmental biology. His research emphasizes the use of genetic and molecular-genetic tools to develop synthetic approaches to interrupting pathogen transmission by mosquitoes. He is an author on more than 170 papers, reviews and policy documents. He was a founding editor of the journal Insect Molecular Biology, and has served on the editorial boards of PLoS Neglected Tropical Diseases, Experimental Parasitology and Entomological Research.
President Larry Millstein called the 2378th meeting of the Society to order at 8:11 p.m. He announced the order of business and welcomed new members. President Millstein presented a summary of the 37th meeting of the Society, held in 1872. The minutes of the previous meeting were read and approved. President Millstein then introduced the speaker for the evening, Anthony A. James, a Distinguished Professor in the Departments of Microbiology & Molecular Genetics and Molecular Biology & Biochemistry at the University of California, Irvine. His lecture was titled “The Mosquito, Synthetic Biology, CRISPR, and Malaria”.
Dr. James began by explaining that malaria has afflicted humans for all of recorded history, with references to deadly recurrent fevers appearing in Mesopotamian cuneiform from 6,000 BC. The famous Greek physician Hippocrates noted the link between swampy waters and fever cycles.
Malaria is a bloodborne infectious disease resulting from a protozoan parasite which is transmitted by mosquitos. Today there are five species of malaria parasite that affect humans, the newest of which crossed over to humans within the last 15 years. The two species most deadly to humans are plasmodium falciparum and plasmodium vivax. Importantly, there are no free-living forms of the parasite; it exists only inside a suitable host. Malaria is only spread by adult female mosquitos, which feed on blood as a source of protein for nutrition and egg production.
Malaria today is endemic in the poorest countries of the world, existing in all tropical and subtropical regions. In 2015, there were 212 million cases worldwide, resulting in approximately 429,000 deaths, 90% of which were in sub-Saharan Africa. Although we have many existing tools that are effective against malaria—including bed nets, spraying, eliminating standing water, and anti-malarial drugs—for various practical, economic, and political reasons, these tools have been insufficient to control malaria in many countries. To date, no effective vaccines have been developed against any protozoan parasite, including malaria.
Dr. James then described a promising new strategy of engineering mosquitos with genes that prevent transmission of the malaria parasite. To do this, scientists first searched for mosquito genes that are activated when the mosquito feeds; they found suitable genes related to digestion. Next, they identified a gene in mice that reacted very strongly to human strains of the malaria parasite, neutralizing it. Splicing this mouse gene into a mosquito creates a transgenic mosquito that express antibodies in the midgut and the salivary gland every time the mosquito feeds, curing the mosquito and eliminating transmission back to humans.
After developing a malaria-immune mosquito, the next task is to distribute these genes throughout the mosquito population. This can be done simply by breeding and releasing millions of mosquitos, but a more effective strategy is to include a “gene drive” in the engineered mosquito’s DNA that ensures that the anti-malarial genes are preferentially inherited when the mosquito breeds, overriding the traditional Mendelian pattern of inheritance.
The creation of a gene drive is possible because of advances in the use of the CRISPR/Cas9 gene editing system, which consists of the Cas9 nuclease, which cuts DNA, plus guide RNA, which targets the cuts to a precise position in the mosquito DNA sequence. When the cell attempts to repair the cut DNA, the most available model for reconstituting the cut section is the lab-created plasmid containing the anti-malarial mouse gene. Moreover, this engineered sequence also contains the coding for the cas9 nuclease and guide RNA, which means that the repaired DNA will copy itself autonomously to the other, unmodified chromosome, creating mosquitos that have a one hundred percent chance of passing on the engineered genes.
Dr. James explained that in addition to the functional parts of the engineered plasmid—the nuclease, guide RNA, and malarial resistance sequence—it also contains a protein for red coloring from a jellyfish that allows engineered mosquitos to be readily distinguished from wild mosquitos.
Dr. James noted that drugs, insecticides, and vaccines all have well-established regulatory pipelines from discovery to development to delivery, but genetics based vector control does not. Nonetheless, Dr. James expressed confidence that carefully constructed, rigorously conducted phased trials will demonstrate to regulators, governments, and communities that genetically-engineered mosquitos are a safe and effective malaria control strategy that, when combined with existing tools, could ultimately enable world health authorities to finally eradicate malaria.
After the conclusion of the lecture, President Millstein invited questions from the audience.
One questioner asked how the mouse gene confers malaria resistance in the mosquito. Dr. James explained that the engineering confers two forms of resistance. First, it creates antibodies that bind to surface proteins that the parasite uses to invade cells, thereby denying it entry into the host cells. The second method simply coats the surface of the parasite in antibodies, preventing it from interacting with the host cells.
Another questioner as why the engineered mosquitos used genes for mouse antibodies and not human antibodies. Dr. James explained that human resistance acts only during the human blood cell stages and does not provide complete protection, so the mouse gene was chosen to target malaria completely and at the point of transmission.
After the question and answer period, President Millstein thanked the speaker, made the usual housekeeping announcements, and invited guests to join the Society. At 9:59 p.m., President Millstein adjourned the 2378th meeting of the Society to the social hour.
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