Lentiviral transduction facilitates RNA interference in the nematode parasite Nippostrongylus brasiliensis

Abstract

Animal-parasitic nematodes have thus far been largely refractory to genetic manipulation, and methods employed to effect RNA interference (RNAi) have been ineffective or inconsistent in most cases. We describe here a new approach for genetic manipulation of Nippostrongylus brasiliensis, a widely used laboratory model of gastrointestinal nematode infection. N. brasiliensis was successfully transduced with Vesicular Stomatitis Virus glycoprotein G (VSV-G)-pseudotyped lentivirus. The virus was taken up via the nematode intestine, RNA reverse transcribed into proviral DNA, and transgene transcripts produced stably in infective larvae, which resulted in expression of the reporter protein mCherry. Improved transgene expression was achieved by incorporating the C. elegans hlh11 promoter and the tbb2 3´-UTR into viral constructs. MicroRNA-adapted short hairpin RNAs delivered in this manner were processed correctly and resulted in partial knockdown of β-tubulin isotype-1 (tbb-iso-1) and secreted acetylcholinesterase B (ache-B). The system was further refined by lentiviral delivery of double stranded RNAs, which acted as a trigger for RNAi following processing and generation of 22G-RNAs. Virus-encoded sequences were detectable in F1 eggs and third stage larvae, demonstrating that proviral DNA entered the germline and was heritable. Lentiviral transduction thus provides a new means for genetic manipulation of parasitic nematodes, including gene silencing and expression of exogenous genes.

Transgenic expression of a T cell epitope in Strongyloides ratti reveals that helminth-specific CD4+ T cells constitute both Th2 and Treg populations

“…Though it is not clear why the C terminal region of the transgene is lost as Hulk develops within its host, there are two, non-mutually exclusive explanations: (a) the transgene, though constitutively expressed, becomes truncated or silenced at the transcript or protein level in the L4 and parasitic adult stages, and (b) the Ss-act-2 promoter does not drive constitutive expression throughout the S. ratti life cycle. The first explanation is consistent with recent data showing that lentiviral-transduced N. brasiliensis worms can retain an mCherry transgene in their genomic DNA throughout ontogeny, but that transcription of this transgene is lost in later, parasitic developmental stages of the worm [45]. Given the immunogenic nature of the 2W1S peptide, there may be selective pressure in hosts for adults to silence or edit the transgene at the transcript or protein level.…”

Transgenesis in parasitic helminths: a brief history and prospects for the future

“…1 ; Table 1 ). Nonetheless, no obvious phenotypic effect after silencing any of the targets ( β-tubulin isotype-1 and secreted acetylcholinesterase B ) were observed in L3 in vitro , possibly due to the modest knockdown of target mRNA achieved and the lack of shRNAmer expression in adult worms (Table 1 ) [ 82 ]. The authors hypothesised that the lack of transcription could be due to chromatin-mediated transcriptional silencing of the viral integration site during parasite development.…”

“…The authors hypothesised that the lack of transcription could be due to chromatin-mediated transcriptional silencing of the viral integration site during parasite development. Further optimisation of the expression cassette is needed to overcome the problem of transcriptional silencing during development [ 82 ].…”

The Wild Worm Codon Adapter: a web tool for automated codon adaptation of transgenes for expression in non-Caenorhabditis nematodes

“…In B. malayi , transfection of infective larvae has been used to deliver reporter plasmids and CRISPR constructs ( Liu et al 2018 , 2020 ). Most recently, lentiviral transduction of infective larvae was used to deliver RNA interference molecules and drive expression of fluorescent reporters in N. brasiliensis ( Hagen et al 2021 ).…”

“…Codon usage bias is believed to regulate the expression of exogenous transgenes as well as endogenous genes ( Redemann et al 2011 ). In non- Caenorhabditis nematodes, expression of exogeneous genes from transgenes is promoted by the use of species-specific codon usage patterns ( Han et al 2020 ; Hagen et al 2021 ).…”