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-8168604-B2
Publication Date
2012-05-01
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 growth of viruses of the Filoviridae family, including treatment of viral infections in mammals such as primates by Ebola and Marburg viruses. The antisense antiviral compounds are morpholino oligonucleotides characterized by a nuclease resistant backbone, 15-40 nucleotide bases, and targeting sequences of at least 15 bases in length that hybridize specifically to target regions including the Ebola virus AUG start site regions of VP24 or VP35, and the Marburg virus AUG start site regions of VP24 or NP.
The problem addressed arises from the lack of effective antiviral therapies for infections caused by (−) RNA viruses, particularly the Filoviridae family such as Ebola and Marburg viruses which cause severe and often lethal hemorrhagic fever in humans. Existing treatments are supportive rather than curative, and effective vaccines or antiviral drugs are unavailable, compounded by difficulties in working with these viruses due to biosafety concerns and their biological properties. There is a need for well-orchestrated, sequence-specific treatments to inhibit viral gene expression, thereby controlling infection and improving therapeutic outcomes.
The solution involves developing antisense oligonucleotide analog compounds that possess properties enabling active or facilitated uptake into infected host cells and stable, sequence-specific binding to viral mRNA target sequences surrounding the AUG start codon. These antisense compounds, particularly phosphorodiamidate morpholino oligomers (PMOs), form stable heteroduplex structures with viral RNA, resist nucleases, and sterically block viral RNA translation, resulting in inhibition of viral replication. The compounds may be conjugated to arginine-rich peptides to enhance cellular uptake, with targeting sequences selected from conserved genomic regions in Ebola and Marburg viruses to maximize efficacy.
Claims Coverage
The claims include one independent claim focusing on a method of treating Marburg virus infection using antisense morpholino oligomers, with several dependent claims specifying structural and targeting features, administration details, and combination therapies. There is one independent claim identified.
Antisense oligomer composition for treatment of Marburg virus infection
A method comprising administration of an antisense oligomer consisting of 12-40 morpholino subunits linked by phosphorous-containing intersubunit linkages that join a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, wherein at least 2 and no more than half of these linkages are positively charged at physiological pH. The antisense oligomer has a targeting sequence forming a heteroduplex with the AUG start-site region of the positive-strand mRNA for Marburg viral protein NP, which inhibits virus production.
Targeting sequence specificity to Marburg virus NP AUG start-site
The targeting sequence forms a heteroduplex with the AUG start-site region defined by SEQ ID NO:13 and comprises complementarity to at least 12 contiguous bases of this sequence.
Optimized oligomer properties for enhanced binding and activity
The antisense oligomer has 15-25 subunits and forms a heteroduplex with a Tm of dissociation of at least 45° C., with morpholino subunits joined by intersubunit linkages of specified chemical structure. Targeting sequences may include complementary bases upstream of the AUG start codon and include between 2-8 piperazine-containing linkages and inosine base-pairing moieties to enhance efficacy.
Combination treatment with second antisense oligomer targeting Marburg virus VP24 AUG start-site
Administration in combination with a second antisense oligomer whose targeting sequence forms a heteroduplex with the AUG start-site region defined by SEQ ID NO:9, with complementarity to at least 12 contiguous bases spanning the AUG start codon, including sequences of at least 14-15 bases complementary to SEQ ID NO:9 or having at least 12 contiguous bases of SEQ ID NO:57, also containing between 2-8 piperazine-containing linkages and at least one inosine base-pairing moiety.
The claims cover administering positively charged morpholino antisense oligomers targeted to specific AUG start site regions of Marburg virus NP and VP24 genes, with defined chemical backbones and sequence properties, alone or in combination, to inhibit virus production in infected subjects.
Stated Advantages
The antisense compounds provide sequence-specific inhibition of filovirus replication, including Ebola and Marburg viruses.
The morpholino oligonucleotide backbone confers nuclease resistance, enhancing stability and efficacy in vivo.
Inclusion of cationic linkages increases antisense activity by 10- to 100-fold compared to uncharged PMOs.
The compounds are actively taken up by mammalian cells, facilitating intracellular delivery without complexing agents.
Combination therapies targeting multiple viral gene start sites increase efficacy and demonstrate synergistic effects.
The antisense treatment protects animals from lethal filovirus infection and induces protective immunity upon viral challenge.
Documented Applications
Treatment of viral infections in mammals, including primates, caused by Ebola virus.
Treatment of viral infections in mammals, including primates, caused by Marburg virus.
Post-exposure therapeutic administration to infected subjects to reduce viral load and mortality.
Use in prophylactic vaccination where pretreatment with the antisense compound followed by viral exposure induces immunity.
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