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CN-122012552-A - Tenecteplase mRNA medicine and application thereof

CN122012552ACN 122012552 ACN122012552 ACN 122012552ACN-122012552-A

Abstract

The invention discloses a tenecteplase mRNA medicament and application thereof. The active ingredient of the medicine provided by the invention is mRNA, and is shown as SEQ ID NO. 5. The invention also protects a tenecteplase recombinant plasmid as shown in SEQ ID NO. 4. The inventor of the invention obtains specific DNA molecules through a series of designs and sequence optimization, synthesizes specific recombinant plasmids through gene synthesis, carries out in vitro transcription on the specific recombinant plasmids, obtains cell supernatant proteins through transfection of different amounts of mRNA and at different time points after transfection, and can effectively express the mRNA of the tenecteplase in different cells of different species through ELISA detection.

Inventors

  • XIONG YONGJIA
  • DU YONG
  • ZHANG WENXUE
  • Chen taibiao
  • XU CHENHUA

Assignees

  • 上海丰华天力通生物医药有限公司

Dates

Publication Date
20260512
Application Date
20241111

Claims (10)

  1. 1. An mRNA molecule is characterized in that the nucleotide sequence of the mRNA comprises a sequence shown as SEQ ID NO.6 or a derivative sequence with the same function as that of the SEQ ID NO.6 by replacing, adding and/or deleting one or more amino acids from the SEQ ID NO.6, and uracil in the mRNA molecule is 1-N-Me-PseudoUTP.
  2. 2. The mRNA molecule of claim 1, wherein the mRNA molecule has an untranslated region added to both the 5 'and 3' ends of the sequence shown in SEQ ID NO.6, preferably, the sequence of the untranslated region at the 5 'end is shown in SEQ ID NO.7 and the sequence of the untranslated region at the 3' end is shown in SEQ ID NO. 8.
  3. 3. The mRNA molecule of claim 1, wherein the mRNA molecule has an mRNA cap structure added to the 5 'end of the sequence shown in SEQ ID NO.6, preferably, the mRNA molecule has a poly-A and/or nucleotide tail added to the 3' end of the sequence shown in SEQ ID NO.6, more preferably, the poly-A has a polymerization degree of 100-300.
  4. 4. The mRNA molecule of claim 1, wherein the nucleotide sequence of the mRNA is SEQ ID NO.5 or a derivative sequence with the same function as SEQ ID NO.5 by replacing, adding and/or deleting one or more amino acids from SEQ ID NO.5, and uracil in the mRNA molecule is 1-N-Me-PseudoUTP.
  5. 5. A DNA molecule encoding the mRNA molecule of claim 1, preferably having a nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO. 3.
  6. 6. A recombinant plasmid comprising the DNA molecule of claim 5, preferably the sequence of said recombinant plasmid is as set forth in SEQ ID NO. 4.
  7. 7. A pharmaceutical composition comprising the mRNA molecule of any one of claims 1-4, the DNA molecule of claim 5, or the recombinant plasmid of claim 6, and a pharmaceutically acceptable carrier.
  8. 8. Use of an mRNA according to any one of claims 1 to 4, a DNA molecule according to claim 5, a recombinant plasmid according to claim 6 or a composition according to claim 7 for the preparation of a tenecteplase mRNA drug.
  9. 9. Use of an mRNA according to any one of claims 1 to 4, a DNA molecule according to claim 5, a recombinant plasmid according to claim 6 or a composition according to claim 7 for the preparation of a thrombolytic drug.
  10. 10. The method for preparing mRNA according to any one of claims 4, wherein a recombinant plasmid comprising a gene encoding the mRNA molecule according to any one of claims 4 is taken, digested with restriction enzyme Bsa I and the linearized plasmid is recovered, and then tenecteplase mRNA is obtained by in vitro transcription.

Description

Tenecteplase mRNA medicine and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to a tenecteplase mRNA medicament and application thereof. Background Tenecteplase is a multi-site variant of the tissue plasminogen activator (tissue plasminogen activator, tPA). Tenecteplase is a thrombolytic drug, is a third generation fibrinolytic agent, and is mainly used for thrombolytic therapy clinically. At present, the tenecteplase is mainly used by injecting tenecteplase protein, Messenger RNA, MESSENGER RNA (mRNA), is transcribed from the template strand of DNA and has the same base sequence as the coding strand of the gene and is complementary to the template strand. mRNA carrying genetic information obtained by gene transcription in eukaryotes consists of an exon encoding a protein and an intron without encoding function arranged at intervals. mRNA directly transcribed from a gene needs to be properly modified, sheared and spliced into mature mRNA before it can be transported into cytoplasm for translation to produce protein. Compared with protein, mRNA 1) can be encapsulated and delivered by liposome, namely, the immunogenicity of the mRNA can be reduced, and the half-life of the mRNA can be enlarged, 2) the mRNA is transcribed in vitro to ensure that the yield of the mRNA is higher and the production time is short, and 3) the mRNA can be quickly modified to adjust the half-life and the immunogenicity characteristics, so that the mRNA is favorable for personalized treatment and emergency adaptation to newly-emerging pathogens. Combines various advantages of mRNA, and has very good development prospect in the development of mRNA drugs. mRNA vaccine drugs against a variety of viral (ebola, zika, influenza) infections and cancers have also been attempted to develop and induce a safe and acceptable effective protective immune response. However, mRNA sequence design is a complex process involving a number of factors, including mRNA stability, translation efficiency, and immunogenicity, and thus limits its wide range of development applications. Disclosure of Invention The invention aims to provide an mRNA drug of tenecteplase, which has high expression in different cell lines, can realize that a small amount of mRNA can achieve a therapeutic effect, and has higher safety. The aim of the invention is achieved by the following technical scheme: The invention provides an mRNA molecule, which is characterized in that the nucleotide sequence of the mRNA comprises a sequence shown as SEQ ID NO.6, or a derivative sequence with the same function as that of the SEQ ID NO.6 by replacing, adding and/or deleting one or more amino acids from the SEQ ID NO.6, and uracil in the mRNA molecule is 1-N-Me-PseudoUTP. In some embodiments, the mRNA molecules of the present invention have an additional untranslated region at both the 5 'and 3' ends of the sequence depicted in SEQ ID NO.6, preferably, the 5 'untranslated region is depicted as SEQ ID NO.7 and the 3' untranslated region is depicted as SEQ ID NO. 8. In some embodiments, the mRNA molecules described herein add an mRNA cap structure at the 5' end of the sequence shown in SEQ ID No. 6. The mRNA cap structure may be one commonly used in the art. In some embodiments, the mRNA molecules of the present invention add a polyadenylation and/or nucleotide-like tail at the 3' end of the sequence shown in SEQ ID No.6, where the polyadenylation or nucleotide-like tail may be conventional in the art, and more preferably, the polyadenylation polymerization degree is 100-300. In some embodiments, the nucleotide sequence of the mRNA of the present invention is the sequence of SEQ ID NO.5, or a derivative sequence of SEQ ID NO.5 with one or more amino acids replaced, added and/or deleted and having the same function as SEQ ID NO.5, and uracil in the mRNA molecule is 1-N-Me-PseudoUTP. The invention also protects the coding gene of the mRNA molecule. In a specific example, the nucleotide sequence of the coding gene is shown in SEQ ID NO.2 or SEQ ID NO. 3. The invention also provides a recombinant plasmid containing the coding gene. The recombinant plasmid can be formed by integrating the coding gene of the present invention into a conventional plasmid, such as pUC57-kan plasmid, according to a conventional method in the art. In a more specific example, the recombinant plasmid is specifically shown in SEQ ID NO. 4. The invention also provides a pharmaceutical composition comprising the mRNA molecule, the DNA molecule or the recombinant plasmid of the invention and a pharmaceutically acceptable carrier. The invention also provides an application of the mRNA molecule, the DNA molecule, the recombinant plasmid or the pharmaceutical composition in preparing a tenecteplase mRNA medicament. The invention also provides an application of the mRNA molecule, the DNA molecule, the recombinant plasmid or the pharmaceutical composition in prepar