Broad dengue neutralization in mosquitoes expressing an engineered antibody

Abstract

With dengue virus (DENV) becoming endemic in tropical and subtropical regions worldwide, there is a pressing global demand for effective strategies to control the mosquitoes that spread this disease. Recent advances in genetic engineering technologies have made it possible to create mosquitoes with reduced vector competence, limiting their ability to acquire and transmit pathogens. Here we describe the development of Aedes aegypti mosquitoes synthetically engineered to impede vector competence to DENV. These mosquitoes express a gene encoding an engineered single-chain variable fragment derived from a broadly neutralizing DENV human monoclonal antibody and have significantly reduced viral infection, dissemination, and transmission rates for all four major antigenically distinct DENV serotypes. Importantly, this is the first engineered approach that targets all DENV serotypes, which is crucial for effective disease suppression. These results provide a compelling route for developing effective genetic-based DENV control strategies, which could be extended to curtail other arboviruses. Author summaryWith limited success of traditional vector control methods to curb dengue infections and more than half of the world's population still at risk, there is a need for novel strategies to reduce its impact on public health. Recent advances in genetic technologies has allowed for precise modifications of mosquito genome to make them resistant to infections, thus breaking the transmission cycle. Here we generated engineered Ae. aegypti mosquitoes PLOS Pathogens | https://doi.efficiently expressing a DENV-targeting single-chain variable fragment (scFv) derived from a previously characterized broadly neutralizing human antibody, which blocked infection and transmission in these mosquitoes. To our knowledge, this is the first example of an engineered transgene capable of rendering Ae. aegypti mosquitoes 100% refractory to all four serotypes of DENV. The engineered mosquitoes, in future, could easily be paired with a gene drive, capable of spreading the transgene throughout wild diseasetransmitting mosquito populations and preventing further DENV transmission. Since a number of diverse and well-characterized antibodies exist against other arboviruses (eg chikungunya and Zika, this work also provides a proof-of-concept principle for developing similar genetic strategies for reducing the impact of these arboviruses.

Programmable RNA Targeting using CasRx in Flies

“…For example, targeting GFP, a non-essential gene, produced the largest number of misexpressed genes as well as the most significant fold change in expression compared to all other gene targets analyzed. Additionally, because Gadd45, a gene involved in cellular arrest and apoptosis in Drosophila melanogaster [46], was also significantly misexpressed in 4 samples (w, N, y, and GFP) (Table S8- 12), it is possible that CasRx cleavage may result in an increased level of misexpressed genes leading to lethality or cellular apoptosis. Moreover, for the off target analysis for most of the target genes less than 300 (1.7%) other genes were misexpressed, however for GFP we found 5880 (33%) of genes misexpressed and it remains unclear whether this is a result of guide specific off-target or simply bystander cleavage (i.e.…”

“…Numerous RNA viruses of global medical importance, such as dengue, Zika, and chikungunya virus, are transmitted by one species of Aedes mosquito. Engineering this mosquito population with virus resistance may be a tool to reduce disease transmission [11]; however, no current technologies have successfully targeted all of these viruses simultaneously [12][13][14][15][16]. RNAtargeting CRISPR systems may provide a platform to reduce the spread of mosquito-borne viruses by targeting viral RNA genomes in a programmable manner.…”

A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines

“…albopictus, which is prevalent in North America and is an emerging vector for certain arboviruses, such as chikungunya virus 2,4,5 . The ability to perform functional genetic studies in mosquitoes and mosquito cells is crucial to our understanding of pro- and anti-viral mosquito host factors and for potential mosquito control strategies 6–8 . Previous genome engineering in mosquitoes has been achieved using transposons 8–11 and a variety of engineered nucleases such as transcription activator-like effector nucleases (TALENs) 12,13 , zinc-finger nucleases (ZFNs) 14–17 ; and homing endonucleases (HEs) 6,18,19 .…”

Negeviruses reduce replication of alphaviruses during co-infection

“…Negeviruses also possess this attribute, having been discovered in numerous mosquito species on 6 continents, along with sandflies and other diverse insect species [19][20][21][22]24]. Insertion of gene-editing scFv sequences into mosquito genomes has also been used to prevent Plasmodium and DENV infection [45,46]. By using CRISPR-Cas9 to insert a scFv targeting Plasmodium, infection was blocked in Anopheles mosquitoes, and gene drive insured the production of the scFv in the offspring.…”