Search

EP-4737574-A2 - POLY-TAILED AND POLY-CAPPED MRNA AND USES THEREOF

EP4737574A2EP 4737574 A2EP4737574 A2EP 4737574A2EP-4737574-A2

Abstract

Disclosed herein are modified mRNAs with poly(A) tails containing one or more additional poly-A tails or 5' caps, which may be made by ligation of nucleic acids onto the 3' terminal end or 5' terminal end of an RNA, respectively. Also provided are compositions comprising one or more modified mRNAs provided herein, and methods of using said compositions for therapeutic or agricultural applications.

Inventors

  • ADITHAM, Abhishek
  • CHEN, HONGYU
  • GUO, Jianting
  • WANG, XIAO

Assignees

  • The Broad Institute Inc.
  • Massachusetts Institute of Technology

Dates

Publication Date
20260506
Application Date
20230118

Claims (15)

  1. A modified mRNA comprising: (i) an open reading frame (ORF) encoding a protein; (ii) a poly-A region, and (iii) a 5' cap region; wherein the poly-A region is 3' to the ORF and comprises 10 or more nucleotides and the 5' cap region is 5' to the ORF; and wherein the 5' cap region comprises two or more 5' caps.
  2. The modified mRNA of claim 1, wherein the modified mRNA comprises a poly-A region comprising one poly-A tail and a 5' cap region comprising two or more 5' caps.
  3. The modified mRNA of claim 1 or 2, wherein the modified mRNA comprises a 5' untranslated region (5' UTR) and a 3' untranslated region (3' UTR), wherein the ORF is between the 5' UTR and the 3' UTR, wherein the 3' UTR is between the ORF and the poly-A region, and wherein the 5' UTR is between the 5' cap region and the ORF.
  4. The modified mRNA of any one of claims 1-3, wherein the modified mRNA is a linear mRNA, wherein the 5' cap region is at the 5' end of the modified mRNA and the poly-A region is at the 3' end of the modified mRNA, or wherein the modified mRNA is a circular mRNA, wherein the poly-A region is between the 3' UTR and the 5' cap region.
  5. The modified mRNA of any one of claims 1-4, wherein the 5' cap region comprises two or more 5' caps, and at least two instances of the 5' caps are covalently linked by a linker.
  6. The modified mRNA of claim 5, wherein: the 5' cap region comprises -(a first 5' cap)-[(a linker)-(a second 5' cap) m1 ] m2 ; each instance of m1 is independently an integer between 1 and 20, inclusive; m2 is an integer between 2 and 10, inclusive; the first 5' cap is covalently linked to the 5' UTR; and the linker is covalently linked to the first 5' cap and to the second 5' cap.
  7. The modified mRNA of claim 6, wherein each instance of m1 is 1 or is independently an integer between 2 and 20, inclusive, and optionally wherein at least one instance of the linker comprises a dendrimer.
  8. The modified mRNA of any one of claims 5-7, wherein at least one instance of the linker is covalently linked to a 5' or 3' nucleotide of the first 5' cap.
  9. The modified mRNA of any one of claims 5-8, wherein at least one instance of the linker is covalently linked to a 5' or 3' nucleotide of the at least one instance of the second 5' cap.
  10. The modified mRNA of any one of claims 1-9, wherein the 5' cap region of the modified mRNA comprises one or more modified nucleotides.
  11. The modified mRNA of any one of claims 1-9, wherein the 5' cap region comprises between 25-500 nucleotides.
  12. The modified mRNA of any one of claims 1-10, wherein the 5' cap region comprises between 1 and 3, between 3 and 5, between 5 and 7, or between 7 and 10 5' caps.
  13. The modified mRNA of any one of claims 1-12, wherein the 5' cap region comprises one or more modified 5' caps.
  14. The modified mRNA of claim 13, wherein the one or more modified 5' caps are selected from a locked nucleic acid (LNA)-modified cap, a 5' cap comprising a 5'-5' triphosphate linkage that is modified by 5'-phosphorothiolate, and a 5' cap comprising a 5'-5' tetraphosphate linkage, and a 5' cap comprising one or more modified nucleotides.
  15. The modified mRNA of any one of claims 1-14, for use in preventing or treating a disease in a subject in need thereof.

Description

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (B119570144WO00-SEQ-JQM.xml; Size: 61,200 bytes; and Date of Creation: January 18, 2023) is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION Messenger RNA (mRNA) technology is an emerging alternative to conventional small molecule, DNA, and protein therapeutics and conventional vaccine approaches because it is potent, programmable, and capable of rapid production of mRNAs with desired sequences. mRNA therapeutics are a rapidly developing field and have been used for the expression of therapeutic proteins, ranging from vascular regeneration factors (e.g., vascular endothelial growth factor A (VEGF-A), GATA Binding Protein 4 (GATA4), Myocyte Enhancer Factor 2C (MEF2C), T-Box Transcription Factor 5 (TBX5), myocardin (MYOCD)) to vaccines for COVID-19, influenza, and Zika virus. Despite recent clinical successes, mRNA therapeutics still faces challenges of instability, toxicity, short-term efficacy, and potential immunological responses. Increasing the stability of mRNAs to enhance their efficacy in vivo remains an important problem that must be solved to increase the feasibility of mRNA therapeutics for clinical applications. SUMMARY OF THE INVENTION Provided herein are modified mRNAs with modified poly-A tails and/or modified 5' caps to improve the stability of the mRNA in cells and in in vitro translation systems, thereby enhancing protein production, as well as methods of making and using such modified mRNAs. Conventional mRNAs comprise poly-A tails which include approximately 100-250 adenosine nucleotides at the 3' end. Poly-A tails can be degraded or removed by cellular exonucleases, which remove 3' nucleotides. Once exonucleases remove the poly-A tail and begin removing nucleotides of the 3' untranslated region (UTR) and/or open reading frame (ORF), the mRNA is unable to be translated into an encoded protein. mRNAs that are more resistant to 3' exonuclease activity are degraded more slowly and are thus more stable, having increased half-lives in cells, and therefore more protein can be produced from a given mRNA molecule. A poly-A tail is also required for binding to the poly-A binding protein (PABP), which enhances translation of mRNA molecules by binding to the eukaryotic initiation factor 4 complex (eIF4G). Conventional mRNAs also include a 5' cap which, similar to the poly-A tail, protects mRNAs from 5' exonucleases and serves as a site for the recruitment of translation machinery. Modified nucleotides containing one or more structural modifications to the nucleobase, sugar, or phosphate linkage of the mRNA can interfere with 3' and 5' exonuclease activity, rendering the mRNA more stable. However, the same structural modifications that inhibit exonucleases can also hinder the ability of enzymes to incorporate these modified nucleotides into the mRNA, making the modified mRNA difficult to produce. Nevertheless, alternate strategies for enhancing the stability of mRNA molecules without significantly impairing the synthesis and/or translation of the mRNAs have been demonstrated. Disclosed herein are strategies for enhancing mRNA stability by either ligating multiple poly-A tails onto the 3' end of a mRNA, and/or ligating multiple 5' caps onto the 5' end. One strategy involves ligating onto the 3' and/or 5' end of an existing mRNA molecule an oligonucleotide comprising one or more nucleotides modified with click chemistry handles. In turn, additional oligonucleotides comprising either 3' poly-A tails or 5' caps are ligated to the one or more modified nucleotides via click chemistry reactions (see FIGs. 1C and 1G). Alternately, the same result may be achieved by ligating onto the 3' and/or 5' end of an existing mRNA molecule an oligonucleotide that is attached to a dendrimer through a click chemistry reaction. In turn, the dendrimer, which is also modified with click chemistry handles, is ligated to additional oligonucleotides comprising either 3' poly-A tails or 5' caps via click chemistry reactions (see FIGs. 1E and 1I). In addition to prolonging mRNA lifetime by protecting existing mRNA molecules from exonuclease activity, thereby reducing degradation of the ORF, these strategies may also enhance translation of mRNAs directly, as each additional poly-A tail or 5' cap ligated to the mRNA can interact with and recruit translation machinery to the modified mRNA. These strategies are amenable with one another, and also with other techniques known for enhancing mRNA stability, including, for example, the introduction of modified nucleotides at the 3' and/or 5' ends of a mRNA, the introduction of modified nucleotides into the 3' and/or 5' untranslated regions (UTRs) of an mRNA, the introduction of exonuclease-resistant sequences into a mRNA, and/or circularization by ligating the terminal ends of a linear mRNA to produce a circular mRNA. Accordingly, the present disclosure provides, in some