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-8030291-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 of their use and production for inhibiting growth of viruses of the Filoviridae family, specifically targeting viral infections in mammals including primates by Ebola and Marburg viruses. The antisense antiviral compounds are morpholino oligonucleotides which possess a nuclease-resistant backbone, contain 15 to 40 nucleotide bases, and include a targeting sequence of at least 15 bases complementary to regions around the AUG start sites of viral proteins VP24 and VP35 of Ebola virus and VP24 and nucleoprotein (NP) of Marburg virus.
The background highlights that minus-strand (−) RNA viruses, including those of the Filoviridae family such as Ebola and Marburg viruses, cause severe diseases with high lethality and there are no effective antiviral therapies available. Existing treatments are mostly supportive and experimental approaches have not yielded consistent success, especially in nonhuman primates. The complexity and danger associated with these viruses have impeded development of therapeutics. Thus, there is a clear need for compounds and methods that can effectively treat or inhibit Filovirus infections.
The summary details that the invention discloses antisense oligonucleotides with nuclease-resistant backbones capable of specifically hybridizing to targeted viral mRNA sequences near the AUG start codons, forming stable heteroduplexes with high melting temperatures, effectively blocking viral gene expression and replication. The compounds may contain a mixture of uncharged and positively charged phosphorodiamidate intersubunit linkages to enhance antisense activity and cellular uptake, and can be conjugated to arginine-rich peptides for improved cellular delivery. Methods for treating or vaccinating subjects against Ebola or Marburg virus by administering therapeutically effective amounts of these compounds are also described.
Claims Coverage
The claims encompass an inventive method and compositions for treating Ebola virus infection utilizing antisense morpholino oligomers with specific structural and functional characteristics. There are multiple inventive features focusing on the structure of the oligomers, their targeting sequences, and administration methods.
Antisense oligomer with positively charged linkages targeting Ebola VP24 AUG start-site region
An antisense oligomer composed of 12-40 morpholino subunits linked by phosphorous-containing intersubunit linkages joining the morpholino nitrogen of one subunit to the 5′ exocyclic carbon of an adjacent subunit, wherein at least 2 and no more than half of the linkages are positively charged at physiological pH. The oligomer includes a targeting sequence which forms a heteroduplex with the AUG start-site region of the positive-strand mRNA for Ebola viral protein 24 (VP24), thereby inhibiting virus production.
Specific targeting to defined Ebola VP24 start-site region sequence
The targeting sequence forms a heteroduplex with the AUG start-site region defined by SEQ ID NO:2 and is complementary to at least 12 contiguous bases of this sequence. The oligomer preferably has 15-25 subunits and forms a heteroduplex with a melting temperature (Tm) of at least 45°C.
Structural composition of morpholino subunit linkages
The morpholino subunits are joined by intersubunit linkages having a defined chemical structure as disclosed in the specification, involving phosphorodiamidate linkages.
Treatment of specific Ebola viral strains
The antisense oligomer targets infections caused by specific viral strains, including Ebola Zaire.
Combination therapy with second antisense oligomer targeting Ebola VP35
The method further includes administering a second antisense oligomer structurally identical but with a targeting sequence complementary to the AUG start-site region of the positive-strand mRNA for Ebola viral protein 35 (VP35).
Specific targeting of VP35 AUG start-site region
The VP35 targeting sequence forms a heteroduplex with the AUG start-site region defined by SEQ ID NO:1 and is complementary to at least 12 contiguous bases of this sequence.
Defined targeting sequences corresponding to SEQ ID NO:34 and NO:22
The targeting sequence for VP24 comprises or consists of SEQ ID NO:34, and the VP35 targeting sequence of the second oligomer consists of SEQ ID NO:22.
Specified number of piperazine-containing intersubunit linkages to enhance antisense activity
Antisense oligomers have between 2-8 piperazine-containing (positively charged) intersubunit linkages in the backbone.
Flexible administration modes of combination therapy
The VP24-targeting and VP35-targeting antisense oligomers can be administered sequentially or concurrently.
The claims collectively define a method of treating Ebola infection using morpholino-based antisense oligomers with specific charged intersubunit linkages and targeting sequences complementary to viral mRNA AUG start-site regions, either as single agents or in combination, with specified structural and sequence features ensuring enhanced efficacy and specificity.
Stated Advantages
The antisense compounds provide highly efficacious antiviral activity against Ebola virus infections both in vitro and in vivo in rodent and non-human primate models.
The compounds exhibit high specificity and strong binding affinity to viral mRNA target regions, leading to inhibition of viral replication.
Inclusion of positively charged intersubunit linkages enhances antisense activity and cellular uptake without significant loss of specificity.
The antisense compounds can protect animals from lethal Ebola virus challenge, promote immune responses that confer protection against rechallenge, and are effective in treating established infections.
The oligomers have nuclease-resistant backbones, improving stability and resistance to enzymatic degradation in biological systems.
Use of arginine-rich peptide conjugates further facilitates cellular uptake of the antisense compounds.
Documented Applications
Treatment of viral infections caused by Ebola and Marburg viruses in mammals, including humans and nonhuman primates.
Therapeutic administration of antisense morpholino compounds to infected subjects to inhibit replication of filoviruses.
Vaccination methods involving pretreatment with antisense compounds followed by exposure to attenuated Ebola virus to induce protective immunity.
Detection and identification of specific Ebola viral strains in subjects through administration of antisense oligomers and analysis of heteroduplex formation in body fluids.
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