Inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide‐conjugated morpholino oligomers

Abstract

Delivery of phosphorodiamidate morpholino oligomers (PMO) into fish cells in vitro and tissues in vivo was examined. Uptake was evaluated by fluorescence microscopy and flow cytometry after treating cultured cells or live rainbow trout with 3' fluorescein-tagged PMO. Arginine-rich peptide conjugated to the 5' end of the PMO markedly enhanced cellular uptake in culture by 8- to 20-fold compared with non-peptide-conjugated PMO as determined by flow cytometry. Enhanced uptake of PMO conjugated to peptide was also observed in tissues of fish treated by immersion. The efficacy of PMO as inhibitors of infectious haematopoietic necrosis virus (IHNV) replication was determined in vitro. Peptide-conjugated PMOs targeting sequences within the IHNV genomic RNA (negative polarity) or antigenomic RNA (positive polarity) significantly inhibited replication in a dose-dependent and sequence-specific manner. A PMO complementary to sequence near the 5' end of IHNV genomic RNA was the most effective, diminishing titre by 97%, as measured by plaque assay and Western blot. These data demonstrate that replication of a negative-stranded non-segmented RNA virus can be inhibited by antisense compounds that target positive polarity viral RNA, or by a compound that targets negative polarity viral RNA.

Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: cocktails and chemistries

“…PMOs are not readily taken up by cultured cells, unless high transfection concentrations are applied, scrape loading is employed [28,29], or the PMOs are coupled to cell penetrating peptides to enhance delivery [30-32]. We have undertaken other comparisons between the 2OMeAOs and the PMOs directed at the mdx mouse nonsense mutation in exon 23 and observed that the PMOs offer much greater potential in vivo [16,17,33].…”

In Vitro Resistance Selection and In Vivo Efficacy of Morpholino Oligomers against West Nile Virus

“…Besides flaviviruses, PPMOs have also previously been reported to inhibit other plus-sense RNA viruses (mouse hepatitis virus [36], severe acute respiratory syndrome coronavirus [35], equine arteritis virus [50], and coxsackievirus B3 [56]) and minus-sense RNA viruses (hematopoietic necrosis virus [1], Ebola virus [15], and influenza virus [17]) in cell culture. Furthermore, PPMOs have previously exhibited potency against Ebola virus (15) and coxsackievirus B3 (56) in mouse models.…”

Inhibition of Multiple Subtypes of Influenza A Virus in Cell Cultures with Morpholino Oligomers

“…PMO can interfere with gene expression by stably duplexing with complementary RNA through Watson-Crick base pairing, thus forming a steric block (13,40). The entry of PMO into cells can be markedly increased by conjugation to arginine-rich peptide (ARP) (1,8,29). Recently, ARP-conjugated PMO (P-PMO) have been shown to produce antiviral activity against several RNA viruses in cell culture (1,8,17,32,48) and Ebola virus both in cell culture and in vivo (9).…”

“…The entry of PMO into cells can be markedly increased by conjugation to arginine-rich peptide (ARP) (1,8,29). Recently, ARP-conjugated PMO (P-PMO) have been shown to produce antiviral activity against several RNA viruses in cell culture (1,8,17,32,48) and Ebola virus both in cell culture and in vivo (9). We describe here the evaluation in cell culture of several antisense P-PMO designed to target critical sequence regions in the FLUAV viral genome RNA (vRNA), cRNA, and/or mRNA.…”