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MRNA AND METHOD FOR PRODUCING SAME, AND APPARATUS FOR PRODUCING PROTEIN AND METHOD FOR PRODUCING PROTEIN

Foreign code F210010468
File No. J1036-02WO
Posted date 2021年7月28日
Country 世界知的所有権機関(WIPO)
International application number 2020JP037706
International publication number WO 2021075293
Date of international filing 令和2年10月5日(2020.10.5)
Date of international publication 令和3年4月22日(2021.4.22)
Priority data
  • 特願2019-188411 (2019.10.15) JP
  • 特願2020-121249 (2020.7.15) JP
Title MRNA AND METHOD FOR PRODUCING SAME, AND APPARATUS FOR PRODUCING PROTEIN AND METHOD FOR PRODUCING PROTEIN
Abstract Provided is a mRNA that is used for protein synthesis, and contains a translated region having a start codon and a stop codon and an untranslated region located on the 5'-terminal side of the start codon, wherein some of phosphate groups in at least the range from the 5'-terminal of the untranslated region to 15 nts on the 3'-terminal side of the start codon have replaced with phosphorothioate groups. This method for producing mRNA comprises a step for preparing a DNA template; a step for preparing unmodified NTPs comprising ATP, GTP, CTP, and UTP and preparing modified NTPs in which phosphate groups of at least one kind of these unmodified NTPs are replaced with the phosphorothioate group; and a step for carrying out a transcription reaction using RNA polymerase and using the DNA template as the template and the unmodified NTPs and modified NTPs as substrates.
Outline of related art and contending technology BACKGROUND ART
It is known that introduction of phosphorothioate groups (phosphorothioate groups: PS) into nucleic acids can confer biological stability. Nucleic acids into which PS has been introduced are used in the molecular structure of antisense nucleic acids and siRNA, and are also used in commercially available oligonucleic acid pharmaceuticals. The modified PS (hereinafter, also referred to as "PS modification") mRNA (hereinafter, also referred to as "PS-mRNA") can be enzymatically prepared using a substrate nucleotide triphosphate (NTPαS) in which PS is introduced at the α-position of triphosphate using a T7 RNA polymerase or an e. coli RNA polymerase.
Although methods for synthesizing NTPαS have already been developed, PS modification is hardly used for mRNA, and only a few reported near 30 years ago (see, e.g., Non-Patent Document 1 ~ 3). These reports relate to protein synthesis using the cell-free translation system of.
Non-Patent Document 1 describes that PS-mRNA consisting of a poly-U sequence was synthesized and polyalanine was synthesized at the efficiency of 45 % of the natural sequence (third paragraph from the last paragraph). In this document, sequences are very limited, and the product cannot be strictly said to be a protein.
In Non-Patent Document 2, PS-mRNA is created by an in-vitro transcription method using T7 RNA polymerase using T7 phage DNA as a template. In this document, the translational activity of PS-mRNA was evaluated in the cell-free translational systems of Thermal Thermophilus and Escherichia coli, and it was reported that PS-mRNA in which a single nucleotide was substituted with NTPαS exhibited at most 2-fold protein synthesis in these cell-free systems than in the cell-free systems (Fig.3).
Non-Patent Document 3 produces PS-mRNA of e. coli dihydrofolate reductase by an in vitro transcription method, and measures the amount of protein synthesis using this as a template (Fig.2). In this paper, it is reported that PS-mRNA in which only one type of base was substituted with NTPαS and PS-mRNA in which all four bases were substituted with NTPαS at the time of transcription were produced, and a protein approximately 2 ~ 3 times that of unsubstituted mRNA (also called "PO-mRNA" hereafter) was synthesized by the former. The inventors speculate that this improvement in synthesis efficiency resulted from the stability of PS-mRNA. However, in this document, it is also reported that PS-mRNA in which both four bases were substituted with NTPαS resulted in a lower protein synthesis amount than that of the unsubstituted (PO-mRNA.
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • ABE Hiroshi
  • ABE Naoko
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS IT JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN WS ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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