Anti-Niemann Pick C1 Single-Stranded Oligonucleotides with Locked Nucleic Acids Potently Reduce Ebola Virus Infection In Vitro

Anne Sadewasser,Erik Dietzel,Sven Michel,Michael Klüver,Markus Helfer,Tamara Thelemann,Richard Klar,Markus Eickmann,Stephan Becker,Frank Jaschinski

doi:10.1016/j.omtn.2019.04.018

Abstract

Ebola virus is the causative agent of Ebola virus disease, a severe, often fatal illness in humans. So far, there are no US Food and Drug Administration (FDA)-approved therapeutics directed against Ebola virus. Here, we selected the host factor Niemann-Pick C1 (NPC1), which has been shown to be essential for Ebola virus entry into host cytoplasm, as a therapeutic target for suppression by locked nucleic acid-modified antisense oligonucleotides. Screening of antisense oligonucleotides in human and murine cell lines led to identification of candidates with up to 94% knockdown efficiency and 50% inhibitory concentration (IC50) values in the submicromolar range. Selected candidate oligonucleotides led to efficient NPC1 protein knockdown in vitro without alteration of cell viability. Furthermore, they did not have immune stimulatory activity in cell-based assays. Treatment of Ebola-virus-infected HeLa cells with the most promising candidates resulted in significant (>99%) virus titer reduction, indicating that antisense oligonucleotides against NPC1 are a promising therapeutic approach for treatment of Ebola virus infection.

Highlights

Ebola virus (EBOV), as well as four other filoviruses—Bundibugyo virus (BDBV), Sudan virus (SUDV), Marburg virus (MARV), and Ravn virus (RAVV)—are causative agents of severe disease in humans, such as severe hemorrhagic fever, and are often associated with high morbidity and mortality rates.[1,2,3] These viruses belong to the family Filoviridae of non-segmented negative-strand RNA viruses and are biosafety level 4 pathogens transmitted by contact with body fluids, fomites, and droplets from infected patients
Selection of antisense oligonucleotides (ASOs) Targeting Host Factor Niemann-Pick C1 NPC1-specific 15, 16- and 17-mer ASOs were selected based on human NCBI reference sequence (Table S1)
ASO length, locked nucleic acid (LNA) modification pattern, and localization of ASO binding sequence on human NPC1 mRNA are depicted in Tables 1 and S1 and Figure 1

Summary

Introduction

Ebola virus (EBOV), as well as four other filoviruses—Bundibugyo virus (BDBV), Sudan virus (SUDV), Marburg virus (MARV), and Ravn virus (RAVV)—are causative agents of severe disease in humans, such as severe hemorrhagic fever, and are often associated with high morbidity and mortality rates.[1,2,3] These viruses belong to the family Filoviridae of non-segmented negative-strand RNA viruses and are biosafety level 4 pathogens transmitted by contact with body fluids, fomites, and droplets from infected patients. Filoviruses are considered a significant threat to public health and global security because of their pandemic potential and the risk of being used as a bioweapon.[1,4,5,6] accelerated efforts in the development of therapeutics is a key objective in the filovirus research community, especially since the 2013–2016 EBOV disease (EVD) epidemic in Western Africa. No vaccines or therapeutic agents with final US Food and Drug Administration (FDA) approval are currently available, and supportive care remains the standard for Ebola virus disease treatment. To reduce EBOV spread and the pandemic risk of the current outbreak in Democratic Republic of the Congo (750 confirmed cases and 449 confirmed deaths, as of February 9, 2019) (https://www.who.int/ebola/situation-reports/drc-2018/en/) use of rVSV-ZEBOV Ebola vaccine, as well as antiviral drugs and antibodies against EBOV, have been temporarily approved (https:// www.who.int/ebola/drc-2018/faq-vaccine/en/, https://www.who.int/ ebola/drc-2018/treatments-approved-for-compassionate-use/en/)

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