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Influenza A viruses constitute a major and ongoing global public health concern. Current antiviral strategies target viral gene products; however, the emergence of drug-resistant viruses highlights the need for novel antiviral approaches. Cleavage of the influenza virus hemagglutinin (HA) by host cell proteases is crucial for viral infectivity and therefore presents a potential drug target. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded-DNA-like antisense agents that readily enter cells and can act as antisense agents by sterically blocking cRNA. Here, we evaluated the effect of PPMO targeted to regions of the pre-mRNA or mRNA of the HA-cleaving protease TMPRSS2 on proteolytic activation and spread of influenza viruses in human Calu-3 airway epithelial cells. We found that treatment of cells with a PPMO (T-ex5) designed to interfere with TMPRSS2 pre-mRNA splicing resulted in TMPRSS2 mRNA lacking exon 5 and consequently the expression of a truncated and enzymatically inactive form of TMPRSS2. Altered splicing of TMPRSS2 mRNA by the T-ex5 PPMO prevented HA cleavage in different human seasonal and pandemic influenza A viruses and suppressed viral titers by 2 to 3 log(10) units, strongly suggesting that TMPRSS2 is responsible for HA cleavage in Calu-3 airway cells. The data indicate that PPMO provide a useful reagent for investigating HA-activating proteases and may represent a promising strategy for the development of novel therapeutics to address influenza infections.

A significant consequence of equine arteritis virus (EAV) infection of horses is persistence of the virus in a variable percentage of infected stallions. We recently established an in vitro model of EAV persistence in cell culture for the purpose of furthering our understanding of EAV biology in general and viral persistence in the stallion in particular. In this study we investigated whether persistently infected HeLa cells could be cured of EAV infection by treatment with an antisense peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) designed to target the 5'-terminal region of the EAV genome. We found that persistently infected HeLa cells passaged three times in the presence of 5-10 microM EAV-specific PPMO produced no detectable virus. The PPMO-cured HeLa cells were free of infectious virus, viral antigen and EAV RNA as measured by plaque assay, indirect immunofluorescence assay and RT-PCR, respectively. Furthermore, when re-challenged with EAV at several passages after discontinuation of PPMO treatments, PPMO-cured HeLa cells were found to be refractory to re-infection and to the re-establishment of viral persistence. While these findings demonstrate that PPMO can be used to eliminate persistent EAV infection in cell culture, the efficacy of PPMO against EAV in vivo remains to be addressed., ((c) 2010 Elsevier B.V. All rights reserved.)

Background: Genital herpes, caused by herpes simplex virus type-2 (HSV-2), is a recurrent, lifelong disease affecting tens of millions of people in the USA alone. HSV-2 can be treated therapeutically with acyclovir (ACV) and its derivatives; however, no treatment can prevent HSV reactivation. Novel topical anti-HSV microbicides are much needed to reduce HSV-2 transmission and to treat primary or reactivated infections, especially for ACV-resistant strains. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are single-stranded DNA analogues that enter cells readily and can reduce target gene expression through steric blockage of complementary messenger RNA (mRNA)., Methods: We investigated the antiviral activities of PPMOs targeted to the translation start-site regions of the mRNA for two HSV-2 immediate early genes, immediate early protein (ICP)0 and ICP27, and two early genes, unique long gene (UL)30 and UL39., Results: In cell cultures, PPMOs targeting ICP0 or ICP27 mRNA were found to be highly effective against two strains of HSV-2, one of which was ACV-resistant. In vivo, daily topical applications of up to 1 mM ICP27 PPMO caused no gross or microscopic damage to the genital tract of uninfected BALB/c mice or cotton rats. Cotton rats receiving topical application of ICP27 PPMO 24 h after HSV-2 inoculation showed a reduction in genital lesions and a 37.5% reduction in mortality at 14 days post-infection. Mice receiving topical application of 100 μM of an ICP27 and ICP0 PPMO combination before HSV-2 inoculation had no detectable viral replication in the genital tract at 3-5 days post-infection., Conclusions: These results demonstrate that topically applied PPMOs hold promise as candidate antiviral microbicides against HSV-2 genital infection.

Alternative therapies are needed for HSV-1 infections in patients refractory to treatment with Acyclovir (ACV) and its derivatives. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded DNA analogues that enter cells readily and reduce target gene expression through steric blockage of complementary RNA. When applied before or soon after infection PPMO targeting the translation-start-site regions of HSV-1 ICP0 or ICP27 mRNA reduced HSV-1 plaque formation by 70-98% in vitro. The ICP0 PPMO also reduced ACV-resistant HSV-1 (strain 615.9) plaque formation by 70-90%, while an equivalent dose of ACV produced only 40-50% inhibition when the treatment was applied between 1 and 3hpi. Seven daily topical treatments of 100microg ICP0 PPMO caused no gross or microscopic damage to the corneas of uninfected mice. Topical application of 10microg ICP0 PPMO to the eyes of HSV-1 infected mice reduced the incidence of eye disease by 37.5-50% compared to controls. This study demonstrates that topically applied PPMO holds promise as an antiviral drug candidate against HSV-1 ocular infection.

Measles virus (MeV) is a highly contagious human pathogen. Despite the success of measles vaccination programs, measles is still responsible for an estimated 245,000 deaths each year. There are currently no antiviral compounds available for the treatment of measles. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are antisense compounds that enter cells readily and can interfere with mRNA function by steric blocking. A panel of PPMO was designed to target various sequences of MeV RNA that are known to be important for viral replication. Five PPMO, targeting MeV genomic RNA or mRNA, inhibited the replication of MeV, in a dose-responsive and sequence-specific manner in cultured cells. One of the highly active PPMO (PPMO 454), targeting a conserved sequence in the translation start site of the mRNA coding for the nucleocapsid protein, inhibited multiple genotypes of MeV. This report provides evidence that PPMO treatment represents a promising approach for developing antiviral agents against measles and other paramyxoviruses.

Background: Porcine reproductive and respiratory syndrome (PRRS) causes extensive economic losses in the swine industry. Current strategies and vaccines to control the disease are inadequate. We previously demonstrated that peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) could potently inhibit PRRS virus (PRRSV) replication in cell cultures. PPMOs are single-stranded DNA analogues containing a modified backbone and cell-penetrating peptide. PPMOs are nuclease-resistant, water-soluble, can enter cells readily and exhibit highly specific binding to complementary RNA. In this study, we examined PPMO-mediated inhibition of PRRSV replication in a primary culture of porcine pulmonary alveolar macrophages (PAMs)., Methods: PAMs were collected from piglets, pre-incubated in culture and infected with PRRSV. Viability, cytopathic effects, virus yield and apoptosis of PAMs in the presence or absence of a PPMO (5UP2) were examined. The 5UP2 PPMO is complementary to a conserved sequence in the 5'-terminal region of the PRRSV genome. The level of several interferon-associated gene products and activity of caspases were monitored., Results: PRRSV infection induced the activity of caspases-3/7, -8 and -9 significantly. Treatment of PAMs with 5UP2 resulted in protection of the cells from PRRSV-induced cell death for at least 7 days and avoided the activation of the caspases evaluated. 5UP2 treatment of PRRSV-infected PAMs also prevented the vigorous induction of interferon-beta and chemokines observed in infected and mock-treated PAMs., Conclusions: PPMO-mediated suppression of PRRSV replication in PAMs was associated with a reduction of apoptotic and inflammatory responses. These results provide further rationale for the development of PPMO 5UP2 as an antiviral to control PRRSV infection.

Objectives: To determine the antiviral activity of phosphorodiamidate morpholino oligomers (PMO) and peptide-conjugated PMO (PPMO) in AG129 mice infected with dengue 2 virus (DENV-2)., Methods: Antisense PMO and PPMO were designed against the 5' terminal region (5'SL) or the 3'-cyclization sequence region (3'CS) of DENV genomic RNA and administered to AG129 mice before and/or after infection with DENV-2. In addition, cell culture evaluations designed to determine optimum PPMO length, and pharmacokinetic and toxicity analysis of PPMO were also carried out., Results: Mock-treated AG129 mice lived for 9-17 days following intraperitoneal (ip) infection with 10(4)-10(6) pfu of DENV-2 (strain New Guinea C). Intraperitoneal administration of 5'SL or 3'CS PPMO before and after DENV infection produced an increase in the average survival time of up to 8 days. Animals receiving only post-infection PPMO treatment did not benefit significantly. Cell culture studies showed that PPMO of 22-24 bases long produced substantially higher DENV titre reductions than did PPMO that were either shorter or longer. Pharmacokinetic and toxicology analysis with non-infected animals showed that nine consecutive once-daily ip treatments of 10 mg/kg PPMO resulted in high concentrations of PPMO in the liver and caused little impact on overall health., Conclusions: The data indicate that PPMO had considerable antiviral efficacy against DENV-2 in the AG129 mouse model and that PPMO treatment early in the course of an infection was critical to extending the survival times of DENV-2-infected mice in the AG129 model system.

The genus Alphavirus contains members that threaten human health, both as natural pathogens and as potential biological weapons. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) enter cells readily and can inhibit viral replication through sequence-specific steric blockade of viral RNA. Sindbis virus (SINV) has low pathogenicity in humans and is regularly utilized as a model alphavirus. PPMO targeting the 5'-terminal and AUG translation start site regions of the SINV genome blocked the production of infectious SINV in tissue culture. PPMO designed against corresponding regions in Venezuelan equine encephalitis virus (VEEV) were likewise found to be effective in vitro against several strains of VEEV. Mice treated with PPMO before and after VEEV infection were completely protected from lethal outcome while mice receiving only post-infection PPMO treatment were partially protected. Levels of virus in tissue samples correlated with animal survival. Uninfected mice suffered no apparent ill-effects from PPMO treatment. Thus, PPMO appear promising as candidates for therapeutic development against alphaviruses.

Members of the genera Enterovirus and Rhinovirus (family Picornaviridae) cause a wide range of human diseases. An established vaccine is available only for poliovirus, and no effective therapy is available for the treatment of infections caused by any pathogenic picornavirus. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded DNA-like antisense agents that readily enter cells. A panel of PPMO was tested for their antiviral activities against various picornaviruses. PPMO targeting conserved internal ribosome entry site (IRES) sequence were highly active against human rhinovirus type 14, coxsackievirus type B2, and poliovirus type 1 (PV1), reducing PV1 titers by up to 6 log(10) in cell cultures. Comparative sequence analysis led us to design a PPMO (EnteroX) targeting 22 nucleotides of IRES sequence that are perfectly conserved across greater than 99% of all human enteroviruses and rhinoviruses. EnteroX reduced PV1 replication in cell culture to an extent similar to that of other IRES-specific PPMO. Resistant PV1 arose in cell cultures after 12 passages in the presence of EnteroX and were found to have two mutations within the EnteroX target sequence. Nevertheless, cPVR transgenic mice treated once daily by intraperitoneal (i.p.) injection with EnteroX before and/or after i.p. infection with 3 x 10(8) PFU (three times the 50% lethal dose) of PV1 had an approximately 80% higher rate of survival than the controls. The viral titer in tissues taken at day 5 postinfection showed that animals in the EnteroX-treated group averaged over 3, 4, and 5 log(10) less virus in the small intestine, spinal cord, and brain, respectively, than the amount in the control animals. These results suggest that EnteroX may have broad therapeutic potential against entero- and rhinoviruses.

Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded nucleic acid-analogue antisense agents that enter cells readily and can reduce gene expression by steric blocking of complementary RNA (cRNA) sequences. Here, we tested a panel of PPMO designed to target conserved sequences in the RNA genome segments encoding polymerase subunits of a highly pathogenic mouse-adapted influenza A virus (SC35M; H7N7). Three PPMO, targeting the translation start site region of PB1 or NP mRNA or the 3'-terminal region of NP viral RNA (vRNA), potently inhibited virus replication in MDCK cells. Primer extension assays showed that treatment with any of the effective PPMO led to markedly reduced levels of mRNA, cRNA and vRNA. Initially, the potential toxicity of a range of intranasally administered PPMO doses was evaluated, by measuring their effect on body weight of uninfected mice. Subsequently, a non-toxic dosing regimen was used to investigate the effect of various PPMO on SC35M infection in a mouse model. Mice administered intranasal treatment of PPMO targeting the PB1-AUG region or NP vRNA, at 3 mug per dose, given once 3 h before and once 2 days after intranasal infection with 10xLD(50) of SC35M, showed a 2 log(10) reduction of viral titre in the lungs and 50 % survival for the 16 day duration of the experiment, whereas the NP-AUG-targeted PPMO treatment resulted in 30 % survival of an otherwise lethal infection. These data suggest that PPMO provide a useful reagent to investigate influenza virus molecular biology and may constitute a therapeutic strategy against highly pathogenic influenza viruses.

Porcine reproductive and respiratory syndrome (PRRS) has been devastating the global swine industry for more than a decade, and current strategies to control PRRS are inadequate. In this study we characterized the inhibition of PRRS virus (PRRSV) replication by antisense phosphorodiamidate morpholino oligomers (PMO). Of 12 peptide-conjugated PMO (PPMO), four were found to be highly effective at inhibiting PRRSV replication in cell culture in a dose-dependant and sequence-specific manner. PPMO 5UP2 and 5HP are complementary to sequence in the 5' end of the PRRSV genome, and 6P1 and 7P1 to sequence in the translation initiation regions of ORF6 and ORF7, respectively. Treatment of cells with 5UP2 or 5HP caused a 4.5log(10) reduction in PRRSV yield, compared to a control PPMO. Combination of 6P1 and 7P1 led to higher level reduction than 6P1 or 7P1 alone. 5UP2, 5HP, and a combination of 6P1 and 7P1 inhibited PRRSV replication in porcine alveolar macrophages and protected the cells from PRRSV-induced cytopathic effect. Northern blot and real-time RT-PCR results demonstrated that the effective PPMO led to a reduction of PRRSV RNA level. 5UP2 and 5HP inhibited virus replication of 10 other strains of PRRSV. Results from this study suggest potential applications of PPMO for PRRS control.

RNA virus infections cause immense human disease burdens globally, and few effective antiviral drugs are available for their treatment. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are nuclease resistant and water-soluble single-stranded-DNA-analogues that can enter cells readily and act as steric-blocking antisense agents through stable duplex formation with complementary RNA. Recently there have been a number of publications documenting sequence-specific and dose-dependent inhibition of non-retroviral RNA virus infections by PPMO in both cell culture and murine experimental systems. PPMO have suppressed viral titers by several orders of magnitude in cell cultures, and have reduced viral replication in and/or increased survivorship of mice experimentally infected with poliovirus, coxsackievirus B3, dengue virus, West Nile virus, Venezuelan Equine encephalitis virus, respiratory syncytial virus, Ebola virus and influenza A virus. Along with evaluating PPMO efficacy and toxicity, these studies also explored PPMO mechanism of action, pharmacologic properties and the generation and characterization of resistant virus. Effective PPMO target sites in viral RNA have included regions of highly conserved sequence thought to be important in the pre-initiation or initiation of translation, or in long-range RNA-RNA interactions involved in viral RNA synthesis. These studies provide guidance for the design of steric-blocking antisense agents against RNA viruses, insights into viral molecular biology and novel strategies for the development of antiviral therapeutics. The purpose of this review is to summarize notable findings from the reports documenting antiviral activity by PPMO, with a focus on the specific regions of viral RNA that provided the most effective targets for PPMO-based inhibition of viral replication.

The cellular uptake of PMOs (phosphorodiamidate morpholino oligomers) can be enhanced by their conjugation to arginine-rich CPPs (cell-penetrating peptides). Here, we discuss our recent findings regarding (R-Ahx-R)(4)AhxB (Ahx is 6-aminohexanoic acid and B is beta-alanine) CPP-PMO conjugates in DMD (Duchenne muscular dystrophy) and murine coronavirus research. An (R-Ahx-R)(4)AhxB-PMO conjugate was the most effective compound in inducing the correction of mutant dystrophin transcripts in myoblasts derived from a canine model of DMD. Similarly, normal levels of dystrophin expression were restored in the diaphragms of mdx mice, with treatment starting at the neonatal stage, and protein was still detecTable 22 weeks after the last dose of an (R-Ahx-R)(4)AhxB-PMO conjugate. Effects of length, linkage and carbohydrate modification of this CPP on the delivery of a PMO were investigated in a coronavirus mouse model. An (R-Ahx-R)(4)AhxB-PMO conjugate effectively inhibited viral replication, in comparison with other peptides conjugated to the same PMO. Shortening the CPP length, modifying it with a mannosylated serine moiety or replacing it with the R(9)F(2) CPP significantly decreased the efficacy of the resulting PPMO (CPP-PMO conjugate). We attribute the success of this CPP to its stability in serum and its capacity to transport PMO to RNA targets in a manner superior to that of poly-arginine CPPs.

We characterize in vitro resistance to and demonstrate the in vivo efficacy of two antisense phosphorodiamidate morpholino oligomers (PMOs) against West Nile virus (WNV). Both PMOs were conjugated with an Arg-rich peptide. One peptide-conjugated PMO (PPMO) binds to the 5' terminus of the viral genome (5'-end PPMO); the other targets an essential 3' RNA element required for genome cyclization (3' conserved sequence I [3' CSI] PPMO). The 3' CSI PPMO displayed a broad spectrum of antiflavivirus activity, suppressing WNV, Japanese encephalitis virus, and St. Louis encephalitis virus, as demonstrated by reductions in viral titers of 3 to 5 logs in cell cultures, likely due to the absolute conservation of the 3' CSI PPMO-targeted sequences among these viruses. The selection and sequencing of PPMO-resistant WNV showed that the 5'-end-PPMO-resistant viruses contained two to three mismatches within the PPMO-binding site whereas the 3' CSI PPMO-resistant viruses accumulated mutations outside the PPMO-targeted region. The mutagenesis of a WNV infectious clone demonstrated that the mismatches within the PPMO-binding site were responsible for the 5'-end PPMO resistance. In contrast, a U insertion or a G deletion located within the 3'-terminal stem-loop of the viral genome was the determinant of the 3' CSI PPMO resistance. In a mouse model, both the 5'-end and 3' CSI PPMOs (administered at 100 or 200 microg/day) partially protected mice from WNV disease, with minimal to no PPMO-mediated toxicity. A higher treatment dose (300 microg/day) caused toxicity. Unconjugated PMOs (3 mg/day) showed neither efficacy nor toxicity, suggesting the importance of the peptide conjugate for efficacy. The results suggest that a modification of the peptide conjugate composition to reduce its toxicity yet maintain its ability to effectively deliver PMO into cells may improve PMO-mediated therapy.

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.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The KSHV replication and transcription activator (RTA) and latency-associated nuclear antigen (LANA) play key roles in activating KSHV lytic replication and maintaining KSHV latency, respectively. Phosphorodiamidate morpholino oligomers (PMO) are similar to short single-stranded DNA oligomers, but possess a modified backbone that confers highly specific binding and resistance to nucleases. In this study, RTA and LANA mRNA in PEL cells were targeted by antisense peptide-conjugated PMO (P-PMO) in an effort to suppress KSHV replication. Highly efficient P-PMO uptake by PEL cells was observed. Treatment of PEL cells with a RTA P-PMO (RP1) reduced RTA expression in a dose-dependent and sequence-specific manner, and also caused a significant decrease in several KSHV early and late gene products, including vIL-6, vIRF-1, and ORF-K8.1A. KSHV viral DNA levels were reduced both in cells and culture supernatants of RP1 P-PMO-treated cells, indicating that KSHV lytic replication was suppressed. Treatment of BCBL-1 cells with P-PMO against LANA resulted in a reduction of LANA expression. Cell viability assays detected no cytotoxicity from P-PMO alone, within the concentration range used for the experiments in this study. These results suggest that RP1 P-PMO can specifically block KSHV replication, and further study is warranted.

Coxsackievirus B3 (CVB3) is a primary cause of viral myocarditis, yet no effective therapeutic against CVB3 is available. Nucleic acid-based interventional strategies against various viruses, including CVB3, have shown promise experimentally, but limited stability and inefficient delivery in vivo remain as obstacles to their potential as therapeutics. We employed phosphorodiamidate morpholino oligomers (PMO) conjugated to a cell-penetrating arginine-rich peptide, P007 (to form PPMO), to address these issues. Eight CVB3-specific PPMO were evaluated with HeLa cells and HL-1 cardiomyocytes in culture and in a murine infection model. One of the PPMO (PPMO-6), designed to target a sequence in the 3' portion of the CVB3 internal ribosomal entry site, was found to be especially potent against CVB3. Treatment of cells with PPMO-6 prior to CVB3 infection produced an approximately 3-log(10) decrease in viral titer and largely protected cells from a virus-induced cytopathic effect. A similar antiviral effect was observed when PPMO-6 treatment began shortly after the virus infection period. A/J mice receiving intravenous administration of PPMO-6 once prior to and once after CVB3 infection showed an approximately 2-log(10)-decreased viral titer in the myocardium at 7 days postinfection and a significantly decreased level of cardiac tissue damage, compared to the controls. Thus, PPMO-6 provided potent inhibition of CVB3 amplification both in cell cultures and in vivo and appears worthy of further evaluation as a candidate for clinical development.

Peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMO) are single-stranded nucleic acid-like antisense agents that can reduce gene expression by sterically blocking complementary RNA sequence. P-PMO are water soluble and nuclease resistant, and they readily achieve uptake into cells in culture under standard conditions. Eight P-PMO, each 20 to 22 bases in length, were evaluated for their ability to inhibit influenza A virus (FLUAV) A/PR/8/34 (H1N1) replication in cell culture. The P-PMO were designed to base pair with FLUAV RNA sequences that are highly conserved across viral subtypes and considered critical to the FLUAV biological-cycle, such as gene segment termini and mRNA translation start site regions. Several P-PMO were highly efficacious, each reducing viral titer in a dose-responsive and sequence-specific manner in A/PR/8/34-infected cells. Two P-PMO, one designed to target the AUG translation start site region of PB1 mRNA and the other the 3'-terminal region of nucleoprotein viral genome RNA, also proved to be potent against several other FLUAV strains, including A/WSN/33 (H1N1), A/Memphis/8/88 (H3N2), A/Eq/Miami/63 (H3N8), A/Eq/Prague/56 (H7N7), and the highly pathogenic A/Thailand/1(KAN-1)/04 (H5N1). The P-PMO exhibited minimal cytotoxicity in cell viability assays. High efficacy by two of the P-PMO against multiple FLUAV subtypes suggests that these oligomers represent a broad-spectrum therapeutic approach against a high percentage of known FLUAV strains.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of a contagious disease characterized by reproductive failure in sows and respiratory disease in piglets. This infectious disease results in significant losses in the swine industry and specific anti-PRRSV drugs are needed. In this study, we evaluated a novel class of antisense compounds, peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMOs), for their ability to suppress PRRSV replication in cell culture. P-PMOs are analogs of single-stranded DNA and contain a modified backbone that confers highly specific binding to RNA and resistance to nucleases. Of six P-PMOs tested, one ('5UP1'), with sequence complementary to the 5'-terminal 21 nucleotides of the PRRSV genome, was found to be highly effective at reducing PRRSV replication in a specific and dose-dependent manner in CRL11171 cells in culture. 5UP1 treatment generated up to a 4.5log reduction in infectious PRRSV yield, while a control P-PMO had no effect on viral titer. Immunofluorescence assay with an anti-PRRSV monoclonal antibody confirmed the titer observations. The sequence-specificity of 5UP1 effect was confirmed in part by a cell-free luciferase reporter assay system, which showed that 5UP1-mediated inhibition of translation decreased if the target-RNA contained mispairings in relation to the 5UP1 P-PMO. Real-time RT-PCR showed that the production of PRRSV negative-sense RNA was reduced if 5UP1 was added to cells at up to 6h post-virus inoculation. Cell viability assays detected no cytotoxicity of 5UP1 within the concentration-range of this study. These results indicate that P-PMO 5UP1 has potential as an anti-PRRSV agent.

Phosphorodiamidate morpholino oligomers (PMO) are a class of uncharged single-stranded DNA analogs modified such that each subunit includes a phosphorodiamidate linkage and morpholine ring. PMO antisense agents have been reported to effectively interfere with the replication of several positive-strand RNA viruses in cell culture. The filoviruses, Marburg virus and Ebola virus (EBOV), are negative-strand RNA viruses that cause up to 90% lethality in human outbreaks. There is currently no commercially available vaccine or efficacious therapeutic for any filovirus. In this study, PMO conjugated to arginine-rich cell-penetrating peptide (P-PMO) and nonconjugated PMO were assayed for the ability to inhibit EBOV infection in cell culture and in a mouse model of lethal EBOV infection. A 22-mer P-PMO designed to base pair with the translation start site region of EBOV VP35 positive-sense RNA generated sequence-specific and time- and dose-dependent inhibition of EBOV amplification in cell culture. The same oligomer provided complete protection to mice when administered before or after an otherwise lethal infection of EBOV. A corresponding nonconjugated PMO, as well as nonconjugated truncated versions of 16 and 19 base residues, provided length-dependent protection to mice when administered prophylactically. Together, these data suggest that antisense PMO and P-PMO have the potential to control EBOV infection and are promising therapeutic candidates.

The filoviruses Marburg virus and Ebola virus (EBOV) quickly outpace host immune responses and cause hemorrhagic fever, resulting in case fatality rates as high as 90% in humans and nearly 100% in nonhuman primates. The development of an effective therapeutic for EBOV is a daunting public health challenge and is hampered by a paucity of knowledge regarding filovirus pathogenesis. This report describes a successful strategy for interfering with EBOV infection using antisense phosphorodiamidate morpholino oligomers (PMOs). A combination of EBOV-specific PMOs targeting sequences of viral mRNAs for the viral proteins (VPs) VP24, VP35, and RNA polymerase L protected rodents in both pre- and post-exposure therapeutic regimens. In a prophylactic proof-of-principal trial, the PMOs also protected 75% of rhesus macaques from lethal EBOV infection. The work described here may contribute to development of designer, "druggable" countermeasures for filoviruses and other microbial pathogens.

The antiviral efficacy of ten antisense phosphorodiamidate morpholino oligomers (PMOs) directed against Equine arteritis virus (EAV), a nidovirus belonging to the family Arteriviridae, was evaluated in mammalian (Vero-E6) cells. Peptide-conjugated PMOs (P-PMOs) supplied in cell culture medium at micromolar concentrations were efficiently taken up by Vero-E6 cells and were minimally cytotoxic. The P-PMOs were designed to base pair to RNA sequences involved in different aspects of EAV amplification: genome replication, subgenomic mRNA synthesis, and translation of genome and subgenomic mRNAs. A novel recombinant EAV, expressing green fluorescent protein as part of its replicase polyproteins, was used to facilitate drug screening. A moderate reduction of EAV amplification was observed with relatively high concentrations of P-PMOs designed to anneal to the 3'-terminal regions of the viral genome or antigenome. To determine if the synthesis of subgenomic mRNAs could be specifically reduced, transcription-regulating sequences essential for their production, but not for the production of genomic RNA, were targeted, but these P-PMOs were found to be ineffective at transcription interference. In contrast, all four P-PMOs designed to base pair with targets in the genomic 5' untranslated region markedly reduced virus amplification in a sequence-specific and dose-responsive manner. At concentrations in the low micromolar range, some of the P-PMOs tested completely inhibited virus amplification. In vitro translation assays showed that these P-PMOs were potent inhibitors of translation. The data suggest that these compounds could be useful as reagents for exploring the molecular mechanics of nidovirus translation and have anti-EAV potential at relatively low concentrations.

The recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV) is a potent pathogen of humans and is capable of rapid global spread. Peptide-conjugated antisense morpholino oligomers (P-PMO) were designed to bind by base pairing to specific sequences in the SARS-CoV (Tor2 strain) genome. The P-PMO were tested for their capacity to inhibit production of infectious virus as well as to probe the function of conserved viral RNA motifs and secondary structures. Several virus-targeted P-PMO and a random-sequence control P-PMO showed low inhibitory activity against SARS coronavirus. Certain other virus-targeted P-PMO reduced virus-induced cytopathology and cell-to-cell spread as a consequence of decreasing viral amplification. Active P-PMO were effective when administered at any time prior to peak viral synthesis and exerted sustained antiviral effects while present in culture medium. P-PMO showed low nonspecific inhibitory activity against translation of nontargeted RNA or growth of the arenavirus lymphocytic choriomeningitis virus. Two P-PMO targeting the viral transcription-regulatory sequence (TRS) region in the 5' untranslated region were the most effective inhibitors tested. After several viral passages in the presence of a TRS-targeted P-PMO, partially drug-resistant SARS-CoV mutants arose which contained three contiguous base point mutations at the binding site of a TRS-targeted P-PMO. Those partially resistant viruses grew more slowly and formed smaller plaques than wild-type SARS-CoV. These results suggest PMO compounds have powerful therapeutic and investigative potential toward coronavirus infection.

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.

Noncharged antisense compounds, such as phosphorodiamidate morpholino oligomers (PMOs), do not readily enter mammalian cells in culture. A simple and effective means for cellular delivery of PMOs is through their conjugation to arginine-rich peptides. Understanding the effect of peptide conjugation on the efficacy, toxicity, and specificity of PMOs is important to the successful application of this antisense delivery method. We investigated the effects of conjugation of arginine-rich peptides to PMO on the thermal stability, efficacy and specificity for targeted RNA of the resulting compound. In vitro translation assays showed that (1) R9F2-PMO generated antisense activity 3-25-fold higher than corresponding nonconjugated PMO, (2) the level of antisense activity enhancement by R9F2-PMO over a corresponding nonconjugated PMO is related to the GC content of the PMO sequence, (3) R9F2 conjugation reduced the minimum length of a PMO required to inactivate a target RNA from 20 bases to 14 bases, and (4) nonspecific effects of R9F2-PMO occur at lower concentrations than corresponding PMO alone. Thermal stability of heteroduplexes of PMO and complementary RNA were increased by conjugation of PMO to R9F2 peptide, likely accounting for the increased specific antisense activity of conjugated over nonconjugated PMO. A cell-culture based assay demonstrated that while conjugation to unnatural peptides increased PMO efficacy without causing nonspecificity at concentrations < or = 10 microM, only L-peptide conjugation retained high specificity at higher concentrations. This study demonstrates that conjugation of PMO to an arginine-rich peptide generally increases the binding affinity of the PMO to complementary RNA and increases its antisense potency. Additionally, it is shown that the enzymatic stability of an L- or unnatural peptide used for PMO conjugation affects the antisense properties of the resulting compound.

Five dengue (DEN) virus-specific R5F2R4 peptide-conjugated phosphorodiamidate morpholino oligomers (P4-PMOs) were evaluated for their ability to inhibit replication of DEN virus serotype 2 (DEN-2 virus) in mammalian cell culture. Initial growth curves of DEN-2 virus 16681 were obtained in Vero cells incubated with 20 microM P4-PMO compounds. At 6 days after infection, a P4-PMO targeting the 3'-terminal nucleotides of the DEN-2 virus genome and a random-sequence P4-PMO showed relatively little suppression of DEN-2 virus titer (0.1 and 0.9 log10, respectively). P4-PMOs targeting the AUG translation start site region of the single open reading frame and the 5' cyclization sequence region had moderate activity, generating 1.6- and 1.8-log10 reductions. Two P4-PMO compounds, 5'SL and 3'CS (targeting the 5'-terminal nucleotides and the 3' cyclization sequence region, respectively), were highly efficacious, each reducing the viral titer by greater than 5.7 log10 compared to controls at 6 days after infection with DEN-2 virus. Further experiments showed that 5'SL and 3'CS inhibited DEN-2 virus replication in a dose-dependent and sequence-specific manner. Treatment with 10 microM 3'CS reduced the titers of all four DEN virus serotypes, i.e., DEN-1 (strain 16007), DEN-2 (16681), DEN-3 (16562), and DEN-4 (1036) viruses by over 4 log10, in most cases to below detectable limits. The extent of 3'CS efficacy was affected by the timing of compound application in relation to viral infection of the cells. The 5'SL and 3'CS P4-PMOs did not suppress the replication of West Nile virus NY99 in Vero cells. These data indicate that further evaluation of the 5'SL and 3'CS compounds as potential DEN virus therapeutics is warranted.

Conjugation of a peptide related to the human immunodeficiency virus type 1 Tat represents a novel method for delivery of antisense morpholino-oligomers. Conjugated and unconjugated oligomers were tested to determine sequence-specific antiviral efficacy against a member of the Coronaviridae, Mouse hepatitis virus (MHV). Specific antisense activity designed to block translation of the viral replicase polyprotein was first confirmed by reduction of luciferase expression from a target sequence-containing reporter construct in both cell-free and transfected cell culture assays. Peptide-conjugated morpholino-oligomers exhibited low toxicity in DBT astrocytoma cells used for culturing MHV. Oligomer administered at micromolar concentrations was delivered to >80% of cells and inhibited virus titers 10- to 100-fold in a sequence-specific and dose-responsive manner. In addition, targeted viral protein synthesis, plaque diameter, and cytopathic effect were significantly reduced. Inhibition of virus infectivity by peptide-conjugated morpholino was comparable to the antiviral activity of the aminoglycoside hygromycin B used at a concentration fivefold higher than the oligomer. These results suggest that this composition of antisense compound has therapeutic potential for control of coronavirus infection.

Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli. PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth. However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent response. A significant improvement was made by covalently joining the peptide (KFF)(3)KC to the end of PMOs. In strains with an intact outer membrane, (KFF)(3)KC-myc PMO inhibited luciferase expression by 63%. A second (KFF)(3)KC-PMO conjugate targeted to lacI mRNA induced beta-galactosidase in a dose-dependent response. The end of the PMO to which (KFF)(3)KC is attached affected the efficiency of target inhibition but in various ways depending on the PMO. Another peptide-lacI PMO conjugate was synthesized with the cationic peptide CRRRQRRKKR and was found not to induce beta-galactosidase. We conclude that the outer membrane of E. coli inhibits entry of PMOs and that (KFF)(3)KC-PMO conjugates are transported across both membranes and specifically inhibit expression of their genetic targets.