Inhibition of SARS-CoV-2 growth in the lungs of mice by a peptide-conjugated morpholino oligomer targeting viral RNA

Alexandra Sakai; Gagandeep Singh; Mahsa Khoshbakht; Scott Bittner; Christiane V Löhr; Randy Diaz-Tapia; Prajakta Warang; Kris White; Luke Le Luo; Blanton Tolbert; Mario Blanco; Amy Chow; Mitchell Guttman; Cuiping Li; Yiming Bao; Joses Ho; Sebastian Maurer-Stroh; Arnab Chatterjee; Sumit Chanda; Adolfo García-Sastre; Michael Schotsaert; John R Teijaro; Hong M Moulton; David A Stein

doi:10.1016/j.omtn.2024.102331

Inhibition of SARS-CoV-2 growth in the lungs of mice by a peptide-conjugated morpholino oligomer targeting viral RNA

Mol Ther Nucleic Acids. 2024 Sep 10;35(4):102331. doi: 10.1016/j.omtn.2024.102331. eCollection 2024 Dec 10.

Authors

Alexandra Sakai¹, Gagandeep Singh^{2

3}, Mahsa Khoshbakht⁴, Scott Bittner⁴, Christiane V Löhr⁴, Randy Diaz-Tapia^{2

3}, Prajakta Warang^{2

3}, Kris White^{2

3}, Luke Le Luo⁵, Blanton Tolbert⁵, Mario Blanco⁶, Amy Chow⁶, Mitchell Guttman⁶, Cuiping Li⁷, Yiming Bao^{7

8}, Joses Ho⁹, Sebastian Maurer-Stroh⁹, Arnab Chatterjee¹, Sumit Chanda¹, Adolfo García-Sastre^{2

3

10

11

12

13}, Michael Schotsaert^{2

3

13

14}, John R Teijaro¹, Hong M Moulton⁴, David A Stein⁴

Affiliations

¹ Scripps Research Institute, La Jolla, CA 92037, USA.
² Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
³ Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
⁴ Department of Biomedical Sciences, Carlson College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA.
⁵ Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA.
⁶ Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
⁷ National Genomics Data Center, China National Center for Bioinformation, Beijing 100101, China.
⁸ University of Chinese Academy of Sciences, Beijing 100049, China.
⁹ GISAID @ A∗STAR Bioinformatics Institute, Singapore 138632, Singapore.
¹⁰ Department of Pathology, Molecular, and Cell-Based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
¹¹ Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
¹² Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
¹³ The Icahn Genomics Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
¹⁴ Marc and Jennifer Lipschultz Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Abstract

Further development of direct-acting antiviral agents against human SARS-CoV-2 infections remains a public health priority. Here, we report that an antisense peptide-conjugated morpholino oligomer (PPMO) named 5'END-2, targeting a highly conserved sequence in the 5' UTR of SARS-CoV-2 genomic RNA, potently suppressed SARS-CoV-2 growth in vitro and in vivo. In HeLa-ACE 2 cells, 5'END-2 produced IC₅₀ values of between 40 nM and 1.15 μM in challenges using six genetically disparate strains of SARS-CoV-2, including JN.1. In vivo, using K18-hACE2 mice and the WA-1/2020 virus isolate, two doses of 5'END-2 at 10 mg/kg, administered intranasally on the day before and the day after infection, produced approximately 1.4 log10 virus titer reduction in lung tissue at 3 days post-infection. Under a similar dosing schedule, intratracheal administration of 1.0-2.0 mg/kg 5'END-2 produced over 3.5 log10 virus growth suppression in mouse lungs. Electrophoretic mobility shift assays characterized specific binding of 5'END-2 to its complementary target RNA. Furthermore, using reporter constructs containing SARS-CoV-2 5' UTR leader sequence, in an in-cell system, we observed that 5'END-2 could interfere with translation in a sequence-specific manner. The results demonstrate that direct pulmonary delivery of 5'END-2 PPMO is a promising antiviral strategy against SARS-CoV-2 infections and warrants further development.

Keywords: K-18-hACE2 mouse model; MT: Oligonucleotides: Therapies and Applications; PPMO; RNA virus; SARS-CoV-2; antisense; antiviral; intranasal delivery; intratracheal delivery; peptide-conjugated morpholino oligomer.