Lentiviral delivery of RNAs to effect robust gene silencing in parasitic nematodes

Nematode parasites (roundworms) cause severe disease in farm animals, in particular sheep and cattle, and are responsible for significant economic losses both in the UK and worldwide. Infection is controlled by treating the animals with drugs, but the roundworms are quickly becoming resistant to all available products, a phenomenon known as multidrug resistance. There is an urgent need to design new drugs or develop other means of disease control such as vaccination to protect and intensify livestock production. This aim is hampered by the inability to genetically manipulate nematode parasites in order to define proteins essential for survival or suppression of the immune system. We have developed a new method to manipulate roundworms using viruses to deliver triggers in order to silence gene expression. In this project, we aim to investigate how this works, define the pathways involved, and optimise the system to achieve long-term stable silencing of genes throughout the parasites. We will use the two most commonly used and easily manipulated laboratory models of gastrointestinal roundworm infection. Refinement of this system will enable others in the field to easily adapt and apply it to their species of choice, and will provide a means of defining new targets for control of nematode infection.

This application aims to establish protocols for robust post-transcriptional gene silencing in the two most widely-used laboratory models of gastrointestinal nematode infection, Nippostrongylus brasiliensis and Heligmosomoides polygyrus. Methods employed to effect RNA interference (RNAi) in parasitic nematodes thus far have largely proven ineffective, and we will use lentiviral vectors to ensure delivery of the RNA trigger. We have shown that N. brasiliensis can be successfully transduced with VSV-G-pseudotyped lentivirus, that viral genomic RNA is reverse transcribed into proviral DNA, and that transgene transcripts are produced in infective larvae for up to 4 days. MicroRNA-adapted short hairpin RNAs delivered in this manner are processed correctly and result in partial knockdown of a target transcript. We aim to refine this system by using lentiviral delivery of double stranded RNAs to instigate stable, potent and systemic knockdown in parasitic nematodes via 22G-RNA formation. The main objectives are to characterise the mode of viral transduction, to ensure that the expression cassette is functional throughout the life cycle of the parasites, to optimise gene silencing by engaging endogenous small RNA pathways, and to determine the effect of silencing key targets on parasite development in vivo. Refinement of this system will enable others in the field to easily adapt and apply it to their species of choice, and will provide a means of defining new targets for control of nematode infection.

Gastrointestinal nematode parasites of livestock are a growing problem both in the UK and worldwide. Low level infections contribute to poor weight gain and milk yield, whereas heavy infections can lead to death. Nematode infection of sheep has been estimated to cost the British industry over £80M/yr, and recent surveillance has highlighted a sharp increase in the incidence of helminth infection in both sheep and cattle. Resistance to anthelmintic drugs is developing rapidly, including multidrug resistance to the three major classes in current use. There is an urgent need to design new drugs or develop other means of disease control such as vaccination to protect and intensify livestock production, but progress in defining new targets is hampered by the inability to genetically manipulate nematode parasites. The aim of this project is to develop and refine a new method for initiating gene silencing in parasitic nematodes.

The proposal directly addresses Animal Health, a key area relating to the BBSRC strategic priorities in 'Agriculture and food security' and 'Bioscience for health'. It is aimed at development of intervention strategies for combating infectious diseases that reduce the health and welfare of livestock both in the UK and worldwide. We are very specifically limiting this application to development of gene silencing in laboratory models, as the system will require systematic and incremental optimisation. By the end of the project we should be in a position to translate the technology to nematode parasites of livestock. Knowledge gained from the study will inform development of new drugs and vaccine development against strongylid nematode parasites, which in turn will ultimately lead to increased economic resilience to livestock diseases and more sustainable food production. The ultimate beneficiaries of this research will therefore be farmers and those involved in the livestock industry, in addition to those outlined in 'Academic beneficiaries'.