TY - JOUR
T1 - Incorporation of noncanonical base Z yields modified mRNA with minimal immunogenicity and improved translational capacity in mammalian cells
AU - Zhang, Meng
AU - Singh, Nilmani
AU - Ehmann, Mary Elisabeth
AU - Zheng, Lining
AU - Zhao, Huimin
N1 - This work was supported by the Steve L. Miller Endowed Chair fund and partly by the U.S. National Institutes of Health ( U54DK107965 , R01GM143723 ) (H.Z.). We thank Dr. Teresa Anne Martin (UIUC) for administrative help with IACUC and IBC protocols. We thank Prof. Cecilia Leal (UIUC, MatSE) for sharing the NanoAssemblr Benchtop equipment. The dynamic light scattering experiment was performed at the Materials Research Laboratory (UIUC). We thank Jamie Leann Ludwig (UIUC, Division of Animal Resources) for training with mouse experiments. We thank Dr. Sandra Kay McMasters for providing cell culture media (UIUC, SCS Cell Media Facility). We thank Dr. Gaurav Sahay and Dr. Milan Gautam (Oregon State University) for helpful suggestions on LNPs preparation. We thank Dr. Marcin Krzysztof Wozniak and Kate Janssen (UIUC, Cytometry and Microscopy to Omics Facility) for assistance with flow cytometry. We thank Dr. Austin Joseph Cyphersmith for help with widefield microscopy. We thank Dr. Chunshuai Huang for help with ChemDraw, Dr. Hengqian Ren and Guanhua Xun for helpful discussions. We thank Dr. Huanyu Qiao (UIUC, College of Veterinary Medicine) for providing the MEF cell line. We thank Dr. Hua Wang and Joonsu Han (UIUC, MatSE) for providing BMDCs. Certain figures in this manuscript were created using BioRender.
This work was supported by the Steve L. Miller Endowed Chair fund and partly by the U.S. National Institutes of Health (U54DK107965, R01GM143723) (H.Z.). We thank Dr. Teresa Anne Martin (UIUC) for administrative help with IACUC and IBC protocols. We thank Prof. Cecilia Leal (UIUC, MatSE) for sharing the NanoAssemblr Benchtop equipment. The dynamic light scattering experiment was performed at the Materials Research Laboratory (UIUC). We thank Jamie Leann Ludwig (UIUC, Division of Animal Resources) for training with mouse experiments. We thank Dr. Sandra Kay McMasters for providing cell culture media (UIUC, SCS Cell Media Facility). We thank Dr. Gaurav Sahay and Dr. Milan Gautam (Oregon State University) for helpful suggestions on LNPs preparation. We thank Dr. Marcin Krzysztof Wozniak and Kate Janssen (UIUC, Cytometry and Microscopy to Omics Facility) for assistance with flow cytometry. We thank Dr. Austin Joseph Cyphersmith for help with widefield microscopy. We thank Dr. Chunshuai Huang for help with ChemDraw, Dr. Hengqian Ren and Guanhua Xun for helpful discussions. We thank Dr. Huanyu Qiao (UIUC, College of Veterinary Medicine) for providing the MEF cell line. We thank Dr. Hua Wang and Joonsu Han (UIUC, MatSE) for providing BMDCs. Certain figures in this manuscript were created using BioRender. H.Z. and M.Z. conceived the project and designed the experiments. M.Z. and M.E.E. conducted the in vitro and cellular experiments. M.Z. performed LNPs encapsulation with help from L.Z. for LNPs characterization. M.Z. and N.S. carried out mouse experiments. M.Z. and H.Z. wrote the paper with inputs from all authors. A provisional patent application has been filed based on this study.
PY - 2023/10/20
Y1 - 2023/10/20
N2 - Chemically modified mRNAs hold great potential for therapeutic applications in vivo. Currently, the base modification scheme largely preserves the canonical Watson-Crick base pairing, thus missing one mode of mRNA modulation by altering its secondary structure. Here we report the incorporation of base Z (2-aminoadenine) into mRNA to create Z-mRNA with improved translational capacity, decreased cytotoxicity, and drastically reduced immunogenicity compared to the unmodified mRNA in mammalian cells. In particular, the A-to-Z substitution renders modified mRNAs less immunogenic than the state-of-the-art base modification N1-methylpseudouridine (m1ψ) in mouse embryonic fibroblast cells. As a proof of concept, we developed a Z-mRNA-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antigen-encoding Z-mRNA elicited substantial humoral and cellular immune responses in vivo in mice, albeit with relatively lower efficacy than the state-of-the-art m1ψ-mRNA. Z-mRNA expands the scope of mRNA base modifications toward noncanonical bases and could offer an advantageous platform for mRNA-based therapeutics where minimal immunogenicity is desired.
AB - Chemically modified mRNAs hold great potential for therapeutic applications in vivo. Currently, the base modification scheme largely preserves the canonical Watson-Crick base pairing, thus missing one mode of mRNA modulation by altering its secondary structure. Here we report the incorporation of base Z (2-aminoadenine) into mRNA to create Z-mRNA with improved translational capacity, decreased cytotoxicity, and drastically reduced immunogenicity compared to the unmodified mRNA in mammalian cells. In particular, the A-to-Z substitution renders modified mRNAs less immunogenic than the state-of-the-art base modification N1-methylpseudouridine (m1ψ) in mouse embryonic fibroblast cells. As a proof of concept, we developed a Z-mRNA-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antigen-encoding Z-mRNA elicited substantial humoral and cellular immune responses in vivo in mice, albeit with relatively lower efficacy than the state-of-the-art m1ψ-mRNA. Z-mRNA expands the scope of mRNA base modifications toward noncanonical bases and could offer an advantageous platform for mRNA-based therapeutics where minimal immunogenicity is desired.
KW - Biochemistry
KW - Biological sciences
KW - Medical biochemistry
UR - http://www.scopus.com/inward/record.url?scp=85170262148&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85170262148&partnerID=8YFLogxK
U2 - 10.1016/j.isci.2023.107739
DO - 10.1016/j.isci.2023.107739
M3 - Article
C2 - 37720088
AN - SCOPUS:85170262148
SN - 2589-0042
VL - 26
JO - iScience
JF - iScience
IS - 10
M1 - 107739
ER -