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

Creators: Sakai, Alexandra¹; Singh, Gagandeep²; Khoshbakht, Mahsa³; Bittner, Scott³; Löhr, Christiane V.³; Diaz-Tapia, Randy²; Warang, Prajakta²; White, Kris²; Luo, Luke Le⁴; Tolbert, Blanton⁴; Blanco, Mario⁵; Chow, Amy⁵; Guttman, Mitchell⁵; Li, Cuiping⁶; Bao, Yiming^{6, 7}; Ho, Joses²; Maurer-Stroh, Sebastian²; Chatterjee, Arnab¹; Chanda, Sumit¹; García-Sastre, Adolfo^{2, 8}; Schotsaert, Michael²; Teijaro, John R.¹; Moulton, Hong M.³; Stein, David A.³

1. Scripps Research Institute
2. Icahn School of Medicine at Mount Sinai
3. Oregon State University
4. Case Western Reserve University
5. California Institute of Technology
6. National Genomics Data Center, China National Center for Bioinformation, Beijing, China, 100101
7. University of Chinese Academy of Sciences
8. Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029

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.

Copyright and License

Acknowledgement

This work was supported partially through the National Institute of Allergy and Infectious Diseases (NIAID) Antiviral Drug Discovery program, award no. U19AI171443, along with grants NIH/NIAID R01AI160706 and NIH/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) R01DK130425 to M.S. The authors thank Dr. Randy Albrecht for BSL3 lab management at the Icahn School of Medicine at Mount Sinai and Dr. John Adelman (Oregon Health & Science University) for a critical reading of the manuscript.

Contributions

D.A.S., H.M.M., J.R.T., A. Chatterjee, S.C., A.G.-S., and M.S. conceptualized the study. M.K. and H.M.M. produced the PPMO. A.S., G.S., S.B., C.V.L., R.D.-T., P.W., K.W., L.L.L., M.B., A. Chow, J.H., C.L., J.R.T., M.S., H.M.M., and D.A.S contributed to data production and analysis. K.W., B.T., M.G., S.M.-S., Y.B., A. Chatterjee, J.R.T., H.M.M., and D.A.S. supervised various aspects of the project. D.A.S. wrote the initial draft and revisions of the paper. All authors reviewed, offered comments on, and approved the manuscript. Funding for this project was acquired by S.C., M.S., and A.G.-S.

Conflict of Interest

D.A.S. and H.M.M are listed as inventors on a US patent application, which includes a PPMO developed in this study, filed by Oregon State University. The H.M.M. laboratory has received research support from Radiation Control Technology, BriSight Biosciences, and Autoimmunity BioSolutions. The A.G.-S. laboratory has received research support from GSK, Pfizer, Senhwa Biosciences, Kenall Manufacturing, Blade Therapeutics, Avimex, Johnson & Johnson, Dynavax, 7 Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N-fold LLC, Model Medicines, Atea Pharma, Applied Biological Laboratories, and Merck outside of the reported work. A.G.-S. has consulting agreements with the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7 Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, Pfizer, and Prosetta outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott and Astrazeneca. A.G.-S. is listed as an inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. None of the support defined above is directly related to the research described in this paper.

Supplemental Material

Document S1. Table S1, Figures S1, and S2.
Document S2. Article plus supplemental information.

Files

1-s2.0-S216225312400218X-main.pdf

Files (9.4 MB)

Name	Size	Download all
1-s2.0-S216225312400218X-main.pdf md5:fdc74576c2c80c27bd348e8380a28dd2	4.1 MB	Preview Download
1-s2.0-S216225312400218X-mmc1.pdf md5:47dd1f6f4d141b67cedfbe37f24de325	562.6 kB	Preview Download
1-s2.0-S216225312400218X-mmc2.pdf md5:a6b223b0f7d4590152911eddbd9b296e	4.7 MB	Preview Download

	All versions	This version
Views	0	0
Downloads	0	0
Data volume	0 Bytes	0 Bytes