Cytidine-containing tails robustly enhance and prolong protein production of synthetic mRNA in cell and in vivo

Cheuk Yin Li; Zhenghua Liang; Yaxin Hu; Hongxia Zhang; Kharis Daniel Setiasabda; Jiawei Li; Shaohua Ma; Xiaojun Xia; Yi Kuang

doi:10.1016/j.omtn.2022.10.003

Cytidine-containing tails robustly enhance and prolong protein production of synthetic mRNA in cell and in vivo

Mol Ther Nucleic Acids. 2022 Oct 12:30:300-310. doi: 10.1016/j.omtn.2022.10.003. eCollection 2022 Dec 13.

Authors

Cheuk Yin Li¹, Zhenghua Liang¹, Yaxin Hu¹, Hongxia Zhang², Kharis Daniel Setiasabda¹, Jiawei Li³, Shaohua Ma³, Xiaojun Xia², Yi Kuang^{1

4}

Affiliations

¹ Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
² State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong 510060, China.
³ Tsinghua-Berkeley Shenzhen Institute, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, Guangdong 518057, China.
⁴ HKUST Shenzhen Research Institute, Shenzhen, Guangdong 518057, China.

Abstract

Synthetic mRNAs are rising rapidly as alternative therapeutic agents for delivery of proteins. However, the practical use of synthetic mRNAs has been restricted by their low cellular stability as well as poor protein production efficiency. The key roles of poly(A) tail on mRNA biology inspire us to explore the optimization of tail sequence to overcome the aforementioned limitations. Here, the systematic substitution of non-A nucleotides in the tails revealed that cytidine-containing tails can substantially enhance the protein production rate and duration of synthetic mRNAs both in vitro and in vivo. Such C-containing tails shield synthetic mRNAs from deadenylase CCR4-NOT transcription complex, as the catalytic CNOT proteins, especially CNOT6L and CNOT7, have lower efficiency in trimming of cytidine. Consistently, these enhancement effects of C-containing tails were observed on all synthetic mRNAs tested and were independent of transfection reagents and cell types. As the C-containing tails can be used along with other mRNA enhancement technologies to synergically boost protein production, we believe that these tails can be broadly used on synthetic mRNAs to directly promote their clinical applications.

Keywords: CNOT complex; MT: Oligonucleotides; Therapies and Applications; mRNA half-life; protein production enhancement; synthetic mRNA; tail sequence.