{"title":"Terminal hairpins improve protein expression in IRES-initiated mRNA in the absence of a cap and polyadenylated tail","authors":"Victor Solodushko, Brian Fouty","doi":"10.1038/s41434-023-00391-4","DOIUrl":null,"url":null,"abstract":"Synthesizing mRNA in vitro is a standard and simple procedure. Adding the 5′ cap and 3′ polyadenylated (poly(A)) tail to make this mRNA functional for use as a vaccine or therapy increases the time and cost of production and usually decreases the yield, however. We designed mRNA that lacked a cap and poly(A) tail but included an internal ribosomal entry site (IRES) to initiate protein translation. To protect the 5′ and 3′ ends of mRNA from exonucleases, we added stable terminal hairpins. When compared against typical mRNA (i.e., mRNA that contained a cap and poly(A) tail but lacked hairpins), expression of the delivered reporter protein in HEK293 cells was similar. Using a triple instead of a single hairpin at each end increased protein expression even more. This method has the potential to simplify the production and reduce the cost of synthesizing exogenous mRNA for use as biologics or vaccines.","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":null,"pages":null},"PeriodicalIF":4.6000,"publicationDate":"2023-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9951143/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Gene Therapy","FirstCategoryId":"3","ListUrlMain":"https://www.nature.com/articles/s41434-023-00391-4","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Synthesizing mRNA in vitro is a standard and simple procedure. Adding the 5′ cap and 3′ polyadenylated (poly(A)) tail to make this mRNA functional for use as a vaccine or therapy increases the time and cost of production and usually decreases the yield, however. We designed mRNA that lacked a cap and poly(A) tail but included an internal ribosomal entry site (IRES) to initiate protein translation. To protect the 5′ and 3′ ends of mRNA from exonucleases, we added stable terminal hairpins. When compared against typical mRNA (i.e., mRNA that contained a cap and poly(A) tail but lacked hairpins), expression of the delivered reporter protein in HEK293 cells was similar. Using a triple instead of a single hairpin at each end increased protein expression even more. This method has the potential to simplify the production and reduce the cost of synthesizing exogenous mRNA for use as biologics or vaccines.
期刊介绍:
Gene Therapy covers both the research and clinical applications of novel therapeutic techniques based on a genetic component. Over the last few decades, significant advances in technologies ranging from identifying novel genetic targets that cause disease through to clinical studies, which show therapeutic benefit, have elevated this multidisciplinary field to the forefront of modern medicine.