Engineering of Dengue Virus Refractoriness in Aedes aegypti and Development of an Underdominant Gene Drive System in Drosophila melanogaster

Citation

Matzen, Kelly Jean (2012) Engineering of Dengue Virus Refractoriness in Aedes aegypti and Development of an Underdominant Gene Drive System in Drosophila melanogaster. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/YNFQ-WM56. https://resolver.caltech.edu/CaltechTHESIS:05192012-130917004

Abstract

Vector-borne diseases have a profound impact on world health. The two most well-known and costly diseases are dengue fever and malaria, both spread by mosquito vectors. In the last decade, many new solutions to halting the spread of these diseases have been sought, including vector-mediated disease suppression. The work presented here seeks to generate alleles to effect this suppression, and engineer a drive system to replace the native population. Additional work on systems to keep engineered organisms genetically isolated from native populations has also been carried out. Initial studies in C. elegans investigated use of the transitive nature of RNAi in this species to genetically isolate one population from another. This type of speciation could be used in plant populations to limit gene flow of engineered crops into local environments.

The next series of studies details work on engineering of refractoriness alleles. Dengue virus has several enzymatic activities that are essential for its replicative cycle, including an RNA-dependednt RNA polymerase (RdRp) responsible for synthesizing both the sense and antisense viral genomes and a protease responsible for several essential cleavages of the viral polyprotein. Artificial substrates for these proteins were created to act as sensors, triggering an apoptotic response when viral infection occurs. Several generations of constructs were tested, but so far no completely functional sensor has been generated.

Lastly, a series of underdominant gene drive architectures were built and tested in Drosophila melanogaster. Initial systems utilized a Drosophila cell death protein, Hid, as toxin, and engineered microRNAs designed to target the Hid proteins as antidote. Two toxin-antidote pairs were mismatched and positioned on separate chromosomes so that an organism carrying both chromosomes survives, but an organism carrying only a single chromosome is unviable. Construction of a proof-of-principle in the eye was successful, but work in essential tissues is ongoing. Systems using engineered microRNAs as toxins and resupply of the native protein as antidote were tested in essential tissues. Testing of many components has contributed to the development of these systems, but a complete system has not yet been constructed.

Thesis Files