Antisense antiviral compounds and methods for treating a filovirus infection
Inventors
Stein, David A. • Iversen, Patrick L. • Bavari, Sina • Weller, Dwight D.
Assignees
Sarepta Therapeutics Inc • US Army Medical Research and Development Command
Publication Number
US-8030292-B2
Publication Date
2011-10-04
Expiration Date
2025-10-31
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Abstract
The invention provides antisense antiviral compounds and methods of their use and production in inhibition of growth of viruses of the Filoviridae family, and in the treatment of a viral infection. The compounds and methods relate to the treatment of viral infections in mammals including primates by Ebola and Marburg viruses. The antisense antiviral compounds are morpholino oligonucleotides having: a) a nuclease resistant backbone, b) 15-40 nucleotide bases, and c) a targeting sequence of at least 15 bases in length that hybridizes to a target region selected from the following: i) the Ebola virus AUG start site region of VP24; ii) the Ebola virus AUG start site region of VP35; iii) the Marburg virus AUG start site region of VP24; or iv) the Marburg virus AUG start site region of NP.
Core Innovation
The invention provides antisense antiviral compounds and methods for inhibiting the growth of viruses of the Filoviridae family and for treating viral infections in mammals, including primates, infected by Ebola and Marburg viruses. The antisense antiviral compounds are morpholino oligonucleotides characterized by a nuclease-resistant backbone, having 15 to 40 nucleotide bases, and possessing a targeting sequence of at least 15 bases in length that hybridizes to a target region selected from the AUG start site regions of Ebola virus VP24 or VP35, or Marburg virus VP24 or NP.
Minus-strand (−) RNA viruses, such as members of the Filoviridae family including Ebola and Marburg viruses, cause serious human illnesses with high lethality and lack effective antiviral drugs or vaccines. Ebola virus infections, particularly Ebola Zaire, have caused severe outbreaks with mortality rates up to 90%, and successful therapies have been hindered by difficulties including biological hazard containment, inadequate understanding of virus pathogenesis, and lack of reagents. Current treatments are largely supportive with limited efficacy in nonhuman primate models. Thus, there is a pressing need for therapeutic compounds and methods that specifically target filoviruses to inhibit viral replication and improve treatment outcomes.
The invention addresses this need by providing antisense oligonucleotide analog compounds that specifically hybridize to the AUG start-site regions of filovirus positive-strand mRNA sequences, thereby inhibiting viral protein translation and replication. The compounds have properties including a nuclease-resistant and substantially uncharged morpholino backbone, which enables active or facilitated uptake by mammalian cells, formation of stable heteroduplexes with viral RNA having melting temperatures above 45°C, and substantial resistance to intracellular nucleases. Antisense compounds optionally contain cationic linkages to enhance activity and may be conjugated to arginine-rich peptides to improve cellular uptake. Methods include administration of these compounds to infected subjects and have demonstrated efficacy in vitro and in vivo against Ebola and Marburg virus infections.
Claims Coverage
The claims include one independent claim defining a method of treating Marburg virus infection using an antisense morpholino oligomer with specific structural and targeting properties.
Antisense oligomer with cationic intersubunit linkages targeting Marburg VP24 AUG start-site region
A method of treating Marburg virus infection by administering an antisense oligomer of 12-40 morpholino subunits linked by phosphorous-containing intersubunit linkages joining morpholino nitrogen to 5′ exocyclic carbon, where at least 2 and no more than half the linkages are positively charged at physiological pH, and the oligomer targets the AUG start-site region of Marburg viral protein 24 mRNA, inhibiting virus production.
Specific sequence targeting Marburg VP24 AUG start-site region defined by SEQ ID NO:9
The targeting sequence forms a heteroduplex with the Marburg VP24 AUG start-site region defined by SEQ ID NO:9, with complementarity of at least 12 contiguous bases, and oligomer length of 15-25 subunits.
Heteroduplex stability with melting temperature of at least 45°C
The heteroduplex formed between the antisense oligomer and viral target RNA has a dissociation melting temperature of at least 45°C.
Oligomer backbone linkage structure
The morpholino subunits are joined by intersubunit linkages in accordance with a specified phosphorodiamidate backbone structure.
Combination therapy with second antisense oligomer targeting Marburg NP
Administration of a second antisense oligomer having the same structural characteristics and targeting the AUG start-site region of Marburg nucleoprotein (NP) mRNA, forming heteroduplex complementary to SEQ ID NO:13.
Sequence specificity and length requirements for NP targeting oligomer
The NP-targeting sequence is complementary to at least 12 contiguous bases, preferably 14-20 bases upstream of the AUG start codon, and the oligomer includes 2-8 piperazine-containing intersubunit linkages and at least one inosine base-pairing moiety.
Administration regimens
The oligomers targeting VP24 and NP can be administered sequentially or concurrently to treat Marburg virus infection.
The inventive claims cover methods of treating Marburg virus infection through administration of morpholino antisense oligomers with cationic backbone linkages targeting specific AUG start-site regions of viral mRNAs, alone or in combination, characterized by sequence specificity, structural features, and heteroduplex stability that result in inhibition of virus production.
Stated Advantages
The antisense compounds effectively inhibit Ebola and Marburg virus replication in vitro and in vivo, demonstrating significant reduction in viral titers and improved survival in rodent and non-human primate models.
They exhibit high specificity and sequence-directed action against viral gene expression with stable heteroduplex formation resistant to nuclease degradation.
The compounds have favorable pharmacokinetic and safety profiles, proven by prior clinical trials of morpholino oligomers.
Infected hosts treated with these compounds develop protective immune responses, allowing for effective vaccination strategies through pretreatment and controlled viral exposure.
Documented Applications
Treatment of viral infections caused by filoviruses, including Ebola and Marburg viruses, in mammals such as humans and non-human primates.
Antisense therapeutic methods involving administration of morpholino oligomers targeting AUG start-site regions of viral mRNAs to inhibit viral replication and improve survival.
Use in prophylactic vaccination strategies by pretreating subjects with antisense compounds prior to viral exposure.
Diagnostic identification of Ebola viral strains by administration of antisense oligomers followed by detection of nuclease-resistant heteroduplexes in body fluids.
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