RESUMEN
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.
Asunto(s)
Antivirales/uso terapéutico , Herpesvirus Humano 1/efectos de los fármacos , Proteínas Inmediatas-Precoces/efectos de los fármacos , Queratitis Herpética/tratamiento farmacológico , Morfolinas/uso terapéutico , Ubiquitina-Proteína Ligasas/efectos de los fármacos , Aciclovir/farmacología , Animales , Antivirales/administración & dosificación , Antivirales/efectos adversos , Secuencia de Bases , Chlorocebus aethiops , Farmacorresistencia Viral , Herpesvirus Humano 1/fisiología , Humanos , Queratitis Herpética/virología , Ratones , Datos de Secuencia Molecular , Morfolinas/efectos adversos , Morfolinas/síntesis química , Morfolinas/química , Morfolinos , Células Vero , Proteínas Virales/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Phosphorodiamidate morpholino oligomers (PMOs) are uncharged nucleic acid-like molecules designed to inactivate the expression of specific genes via the antisense-based steric hindrance of mRNA translation. PMOs have been successful at knocking out viral gene expression and replication in the case of acute viral infections in animal models and have been well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents that may be useful for combating filoviral infections. We have previously shown that mice treated with a PMO whose sequence is complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebola virus challenge. In the present study, we report on the abilities of two additional VP24-specific PMOs to reduce the cell-free translation of a VP24 reporter, to inhibit the in vitro replication of Ebola virus, and to protect mice against lethal challenge when the PMOs are delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess the enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of various lengths and compositions, positioning of conjugated peptides to either the 5' or the 3' terminus, and the conferring of charge modifications by the addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and mice during lethal Ebola virus challenge.
Asunto(s)
Antivirales , Ebolavirus/efectos de los fármacos , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Morfolinas , Oligonucleótidos Antisentido , Animales , Antivirales/química , Antivirales/farmacología , Antivirales/uso terapéutico , Chlorocebus aethiops , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ebolavirus/genética , Ebolavirus/fisiología , Femenino , Fiebre Hemorrágica Ebola/virología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Morfolinas/química , Morfolinas/farmacología , Morfolinas/uso terapéutico , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/uso terapéutico , Conejos , Relación Estructura-Actividad , Resultado del Tratamiento , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antivirales/uso terapéutico , Dengue/tratamiento farmacológico , Morfolinas/uso terapéutico , Oligonucleótidos Antisentido/uso terapéutico , Animales , Antivirales/efectos adversos , Antivirales/farmacocinética , Antivirales/farmacología , Peso Corporal , Virus del Dengue/efectos de los fármacos , Virus del Dengue/genética , Inyecciones Intraperitoneales , Hígado/química , Ratones , Morfolinas/efectos adversos , Morfolinas/farmacocinética , Morfolinas/farmacología , Morfolinos , Oligonucleótidos Antisentido/efectos adversos , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Antisentido/farmacología , Plasma/química , Análisis de Supervivencia , Ensayo de Placa ViralRESUMEN
L1R, a myristylated late gene product of vaccinia virus, is essential for formation of infectious intracellular mature virions (IMV). In its absence, only viral particles arrested at an immature stage are detected and no infectious progeny virus is produced. Previous studies have shown that the L1R protein is exclusively associated with the IMV membrane and that myristylation is required for correct targeting. The L1R protein contains six cysteine amino acid residues that have all been shown to participate in intramolecular disulphide bonds. However, it was not clear what role, if any, the disulfide bonds play in the membrane topology of the L1R protein. To address this question, a comprehensive library of L1R mutants in which the cysteine residues have been mutated to serine (either individually or in combination) were tested for their ability to rescue a L1R conditional lethal mutant virus under non-permissive conditions. Much to our surprise, we determined that C57 was not essential for production of infectious IMV. These results suggest that protein disulphide isomerases may be involved in reorganization of disulfide bonds within the L1R protein.