Antiviral Effects of Antisense Morpholino Oligomers in Murine Coronavirus Infection Models

Abstract

The recent emergence of novel pathogenic human and animal coronaviruses has highlighted the need for antiviral therapies that are effective against a spectrum of these viruses. We have used several strains of murine hepatitis virus (MHV) in cell culture and in vivo in mouse models to investigate the antiviral characteristics of peptide-conjugated antisense phosphorodiamidate morpholino oligomers (P-PMOs). Ten P-PMOs directed against various target sites in the viral genome were tested in cell culture, and one of these (5TERM), which was complementary to the 5 terminus of the genomic RNA, was effective against six strains of MHV. Further studies were carried out with various arginine-rich peptides conjugated to the 5TERM PMO sequence in order to evaluate efficacy and toxicity and thereby select candidates for in vivo testing. In uninfected mice, prolonged P-PMO treatment did not result in weight loss or detectable histopathologic changes. 5TERM P-PMO treatment reduced viral titers in target organs and protected mice against virus-induced tissue damage. Prophylactic 5TERM P-PMO treatment decreased the amount of weight loss associated with infection under most experimental conditions. Treatment also prolonged survival in two lethal challenge models. In some cases of high-dose viral inoculation followed by delayed treatment, 5TERM P-PMO treatment was not protective and increased morbidity in the treated group, suggesting that P-PMO may cause toxic effects in diseased mice that were not apparent in the uninfected animals. However, the strong antiviral effect observed suggests that with further development, P-PMO may provide an effective therapeutic approach against a broad range of coronavirus infections.

Inhibition of Foot-and-Mouth Disease Virus Infections in Cell Cultures with Antisense Morpholino Oligomers

“…It is most likely that the cytotoxicity generated by all of the PPMOs in this study at concentrations over 5 M was due to the peptide component of the PPMO. We note the recent development of an alternate arginine-rich peptide (often named P7) (5,9,42) shown to be equivalent to the R 9 F 2 peptide used in this study in its ability to deliver PMO into cells yet less toxic (1), more stable (27), and less affected by serum (9). Clearly, improvements of this nature will contribute to the utility of PPMO technology.…”

“…(B) Effects of subsequent PPMO treatment on WT and PPMO-treated passage 5 R-AUG2/p5 and R-5D/p5 viruses. The inhibition value in the presence of PPMO is relative to that of the mock-treated control in each case and was calculated as described in the legend to [1][2][3][4][5][6][7] P5 (clones 1-7) 1122, 1126-1128, 1136-1138 [9][10] P5 (clones 9, 10) 1123, 1127, 1137…”

“…It has been demonstrated that PMO efficacy in a cell-free system (27), in cell cultures (24,26), and in vivo (13) can be increased considerably by conjugation of arginine-rich peptides to the PMO 5Ј terminus (creating peptide-conjugated PMOs [PPMOs]). PPMOs have demonstrated effective and specific suppression of several RNA viruses in cell cultures (9,13,14,20,28,40) and against Ebola virus (13) and mouse hepatitis virus (5), both in cell cultures and in vivo. Recently, Yuan et al (42) found that a PPMO designed against IRES sequence reduced coxsackievirus B3 production in cell cultures and in the heart tissues of infected mice.…”

A Morpholino Oligomer Targeting Highly Conserved Internal Ribosome Entry Site Sequence Is Able To Inhibit Multiple Species of Picornavirus

“…PMO have been conjugated to various cell-penetrating peptides (CPPs) in order to enhance both PMO uptake into cells (14,33,63) and antisense efficacy (35). Peptide PMO (PPMO) have demonstrated considerable efficacy against a number of RNA viruses (14,19,27,36,40,54), including foot-and-mouth disease virus (53) in cell cultures and against Ebola virus (17), West Nile virus (13), murine coronaviruses (9), and CVB3 (63) in both cell culture and animal models.…”

Inhibition of Intracellular Growth of Salmonella enterica Serovar Typhimurium in Tissue Culture by Antisense Peptide-Phosphorodiamidate Morpholino Oligomer

“…Recently we reported that a conjugate of a PMO and the membrane-penetrating peptide (RXR) 4 XB (X is 6-aminohex-anoic acid, and B is ␤-alanine) was effective in delivering antisense PMOs into Escherichia coli (25). PMO conjugates of (RXR) 4 XB have been shown previously to enter mammalian cells, where they can modulate gene expression (4,7,10,24,36). These results suggest that PMO-(RXR) 4 XB conjugates have potential for use against intracellular bacteria.…”