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WO-2026093322-A1 - IMPROVED DNA CONSTRUCTS FOR MANUFACTURING MESSENGER RNA BY IN VITRO TRANSCRIPTION

WO2026093322A1WO 2026093322 A1WO2026093322 A1WO 2026093322A1WO-2026093322-A1

Abstract

The present disclosure provides methods and compositions for improving the in vitro transcription (IVT) of messenger RNA (mRNA) using SP6, T7, or KP34 RNA polymerase. In particular, the present disclosure provides in vitro transcription templates with optimized nucleic acid sequences specific for use with SP6, T7, or KP34 RNA polymerase that improve mRNA transcript yield and/or reduce amounts of dsRNA.

Inventors

  • ALLOUCHE, Delphine
  • FRAYSSE, Sophie
  • LEGASTELOIS, ISABELLE

Assignees

  • SANOFI PASTEUR INC.

Dates

Publication Date
20260507
Application Date
20251028
Priority Date
20241028

Claims (16)

  1. 1. A DNA construct comprising the nucleic acid sequence 5’-Pi- ATTTAGGX1GACACTATA-P2-C-3’, wherein: Pi is an upstream promoter region; Xi is selected from G, A, or T; P2 is a downstream promoter region that comprises the transcriptional start site of a messenger RNA (mRNA) transcript and consists of GX2A-S1, wherein: X2 is selected from A or G; and Si is selected from GAGGA, CTGGTGGA, GGGAGGTAG, GAGAGAATT, or TACAAGCTT and is optional; or the 3’ terminal C is the first nucleotide of the 5’ untranslated region (5’ UTR).
  2. 2. The DNA construct of claim 1, wherein the transcriptional start site is GGA or GAA.
  3. 3. The DNA construct of claim 1 or 2, wherein the sequence located immediately downstream of P2-C is AGATCGCC.
  4. 4. The DNA construct of any one of the preceding claims, wherein Pi is 18 nucleotides upstream of the transcriptional start site.
  5. 5. The DNA construct of any one of the preceding claims, wherein Pi is selected from the group consisting of 5’-CTCGCGGTCTTTAATTGCCT-3’ (SEQ ID NO: 3), 5’- TTATGTATCATACACATACG-3’ (SEQ ID NO: 4), 5’- TGGACAAATCTGTGTCTCTT-3’ (SEQ ID NO: 5) and 5’- GCACGTCGCCGCGCAGGTATGGCTCGCGGTCTTTAATTGCCT-3’ (SEQ ID NO: 147).
  6. 6. The DNA construct of any one of the preceding claims, wherein: (a) Si is GAGGA, optionally wherein X2 is A; or (b) Si is CTGGTGGA and X 2 is A; or (c) Si is GGGAGGTAG and X 2 is A; or (d) Si is GAGAGAATT and X 2 is A. 73 Sanofi Ref: PAT24103-WO-PCT
  7. 7. The DNA construct of any one of claims 1-5, wherein Pi is 5’- CTCGCGGTCTTTAATTGCCT-3’ (SEQ ID NO: 3) and Xi is T or A.
  8. 8. The DNA construct of any one of claims 1-5, wherein Pi is 5’- TTATGTATCATACACATACG-3’ (SEQ ID NO: 4) and Xi is T or A.
  9. 9. The DNA construct of any one of claims 1-5, wherein Pi is 5’- TGGACAAATCTGTGTCTCTT-3’ (SEQ ID NO: 5) and Xi is G or A.
  10. 10. The DNA construct of any one of claims 1-5, wherein Pi is 5’- GCACGTCGCCGCGCAGGTATGGCTCGCGGTCTTTAATTGCCT-3’ (SEQ ID NO: 147) and Xi is T.
  11. 11. The DNA construct of any one of the preceding claims, wherein the nucleic acid sequence is selected from the group consisting of: (a) 5 ’ -CTCGCGGTCTTTAATTGCCT ATTT AGGTGAC ACT AT AGGAC-3 ’ (SEQ ID NO: 6); (b) 5’- TTATGTATCATACACATACGATTTAGGTGACACTATAGAAGAGGAC-3’ (SEQ ID NO: 7); (c) 5’- TGGACAAATCTGTGTCTCTTATTTAGGGGACACTATAGAAGAGGAC-3’ (SEQ ID NO: 8); (d) 5’- TTATGTATCATACACATACGATTTAGGAGACACTATAGAAGAGGAC-3’ (SEQ ID NO: 9); (e) 5’- CTCGCGGTCTTTAATTGCCTATTTAGGAGACACTATAGAAGAGGAC-3’ (SEQ ID NO: 10); (f) 5’- CTCGCGGTCTTTAATTGCCTATTTAGGTGACACTATAGAAGGGAGGTAG-3’ (SEQ ID NO: 155); 74 Sanofi Ref: PAT24103-WO-PCT (g) 5 ’ TGGAC AAATCTGTGTCTCTT ATTTAGGGGAC ACT ATAGAAGAGA GAATT-3’ (SEQ ID NO: 156); and (h) 5’- GCACGTCGCCGCGCAGGTATGGCTCGCGGTCTTTAATTGCCTATTTAGGTGACA CTATAGAAGGGAGGTAG-3’ (SEQ ID NO: 215).
  12. 12. A method for manufacturing messenger RNA (mRNA) by in vitro transcription (IVT) comprising: (a) providing the DNA construct of any one of the preceding claims; (b) contacting the DNA construct with an SP6 RNA polymerase under conditions suitable for obtaining mRNA transcripts.
  13. 13. The method of claim 12, wherein the amount of double-stranded RNA (dsRNA) comprised in the mRNA transcripts obtained in step b) is decreased and/or the mRNA transcript yield is increased relative to mRNA transcripts obtained with a reference DNA construct comprising a nucleic acid sequence 5’-ATTTAGGTGACACTATAGGAC-3’ (SEQ ID NO: 2) without an upstream promoter region Pi, wherein the 3’ terminal four nucleic acids of the nucleic acid sequence comprise the transcriptional start site of an mRNA transcript.
  14. 14. The method of claim 13, wherein the amount of dsRNA comprised in the mRNA transcripts obtained in step b) is at least 25%, 30% or 35% lower relative to the mRNA transcripts obtained with the reference DNA construct, optionally wherein the amount of dsRNA is determined by ELISA using antibodies J2 and KI.
  15. 15. The method of any one of claims 12-14, wherein the mRNA transcript yield is increased by at least 15% or 20% relative to the mRNA transcript yield obtained with the reference DNA construct, optionally wherein the mRNA transcript yield is determined by UV spectroscopy at 260 nm.
  16. 16. The method of any one of claims 12-15, further comprising a step of purifying the mRNA transcripts obtained in step b) from the SP6 RNA polymerase, optionally wherein the step of purifying the mRNA transcripts involves a method other than (i) cellulose 75 Sanofi Ref: PAT24103-WO-PCT chromatography, and/or (ii) high-performance chromatography (HPLC) with a buffer system comprising triethylammonium acetate and/or acetonitrile. 76

Description

Sanofi Ref: PAT24103-WO-PCT IMPROVED DNA CONSTRUCTS FOR MANUFACTURING MESSENGER RNA BY IN VITRO TRANSCRIPTION RELATED APPLICATIONS [0001] The present application claims priority from European patent application no. 24306813.7, filed October 28, 2024, the contents of which are herein incorporated by reference in their entirety. SEQUENCE LISTING [2] The present specification makes reference to a Sequence Listing, submitted electronically as an .xml file name “PAT24103-WO-PCT_SL.xml” on October 28, 2025. The .xml file was generated on October 21, 2025, and is 192 KB in size. The entire contents of the sequence listing are herein incorporated by reference. FIELD OF THE INVENTION [3] The present invention relates to improved DNA constructs for the manufacturing of messenger RNA (mRNA) by in vitro transcription (IVT). In particular, the invention provides promoter sequences that are optimized for use with SP6 RNA polymerase, T7 RNA polymerase, or KP34 RNA polymerase. BACKGROUND OF THE INVENTION [4] Messenger RNA (mRNA) is becoming increasingly important as a therapeutic agent. mRNA therapy can be used to restore normal levels of an endogenous protein or provide an exogenous therapeutic protein (e.g., a vaccine antigen or antibody) without permanently altering the genome sequence or entering the nucleus of the cell. mRNA therapy takes advantage of the cell’s own protein production and processing machinery to express a therapeutic peptide, polypeptide, or protein, is flexible to tailored dosing and formulation, and is broadly applicable to any disease or condition that is treatable through the provision of an exogenous protein. [5] The process of manufacturing mRNA for use in therapy typically involves the in vitro transcription (IVT) of mRNA from a DNA template using a phage-derived DNA- dependent RNA polymerase. The DNA template can be prepared by standard molecular Sanofi Ref: PAT24103-WO-PCT biology techniques. For example, a nucleic acid sequence encoding a peptide, polypeptide, or protein of interest may be cloned into a multi-copy plasmid which is then propagated in Escherichia coli (E. coll) and purified for use as a template in an IVT reaction. [6] A synthesis process using a phage-derived DNA-dependent RNA polymerase commonly yields transcriptional by-products in addition to the desired mRNA transcripts. For example, T7 RNA polymerase forms double-stranded RNA (dsRNA) during IVT. Although generally producing less dsRNA than T7 RNA polymerase, SP6 RNA polymerase also forms dsRNA (see, e.g., WO 2022/082001 and WO 2018/157153). The formation of dsRNA is undesirable since it leads to inefficient translation of the administered mRNA product and results in the induction of cytokines, eliciting an interferon (IFN)-mediated inflammatory immune response. [7] As dsRNA is highly immunogenic, it is desirable to eliminate or greatly reduce the amount of dsRNA from the in vitro transcribed mRNA for many reasons including, for example, to limit cytokine induction and reactogenicity in vivo and to avoid time-consuming and expensive purification of the mRNA after in vitro transcription (IVT). Moreover, it is also desirable to maximize mRNA yield to decrease production costs. Therefore, a need remains to improve existing methods for the manufacturing of mRNA by IVT. SUMMARY OF THE INVENTION [8] The present invention is based on the discovery that optimization of the nucleotide sequences 5’- and 3 ’-adjacent to the core promoter utilized by phage-derived DNA-dependent RNA polymerases such as SP6, T7, and KP34 can improve messenger RNA (mRNA) transcript yield while also decreasing the amount of double-stranded RNA (dsRNA) present that is produced during in vitro transcription (IVT). [9] In a first aspect, the invention relates to a DNA construct (e.g., an IVT template for use with SP6 RNA polymerase) comprising the nucleic acid sequence 5’-Pi- ATTTAGGXIGACACTATA-P2-C-3’ (5’-Pi-[SEQ ID NO: l]-P2-C-3’), wherein Pi is an upstream promoter region, Xi is selected from G, A, or T, P2 is a downstream promoter region that comprises the transcriptional start site of a messenger RNA (mRNA) transcript and consists of GX2A-Si, wherein X2 is selected from A or G, Si is GAGGA or CTGGTGGA and is optional, and wherein the 3’ terminal C is the first nucleotide of the 5’ untranslated region Sanofi Ref: PAT24103-WO-PCT (5’ UTR). In some embodiments, the transcriptional start site of the mRNA transcript is GGA. In some embodiments, the transcriptional start site of the mRNA transcript is GAA. In some embodiments, the sequence located immediately downstream of the 3’ terminal C is AGATCGCC. The DNA construct can be linear or linearized. [10] In this first aspect, the amount of dsRNA comprised in the mRNA transcripts obtainable with the DNA construct when used with SP6 RNA polymerase as a template for IVT is decreased and/or the mRNA transcript yield is increased relative to mRNA transcripts obtainable with a reference DNA construct compris