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EP-4735465-A1 - ETHYL MODIFIED RNA CAPS AND METHODS OF USE

EP4735465A1EP 4735465 A1EP4735465 A1EP 4735465A1EP-4735465-A1

Abstract

Provided herein are compounds that are cap analogs for polynucleotides that bear a N-ethylguanosine moiety, e.g., RNA molecules, such as mRNA molecules. Also provided are capped polynucleotides, e.g., capped RNA molecules, such as capped mRNA molecules, wherein the 5' end of the RNA molecule comprises a cap analog disclosed herein, drug products comprising the capped RNA molecules, methods for making capped polynucleotides disclosed herein, and kits for making the capped polynucleotides. (I)

Inventors

  • MCKINLAY, Colin James
  • WOO-DERLETH, WAN LU

Assignees

  • Nutcracker Therapeutics, Inc.

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1 . A compound of Formula (I), or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof: wherein X is 0 or S; R 1 is OH or a polynucleotide, and R 2 is H or Me.
  2. 2. The compound, stereoisomer, tautomer, or salt of claim 1 , wherein X is 0.
  3. 3. The compound, stereoisomer, tautomer, or salt of claim 1 , wherein X is S.
  4. 4. The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 3, wherein R 1 is OH.
  5. 5. The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 4, wherein R 2 is H.
  6. 6. The compound, stereoisomer, tautomer, or salt of any one of claims 1 to 4, wherein R 2 is Me.
  7. 7. A compound as shown in Table 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.
  8. 8. An RNA molecule, wherein the 5' end of the RNA molecule comprises the compound, stereoisomer, tautomer, or salt of any one of claims 1 to 7.
  9. 9. The RNA molecule of claim 8, wherein the RNA molecule is mRNA.
  10. 10. The RNA molecule of claim 9, wherein the mRNA encodes for one or more immunomodulatory agents (e.g., a checkpoint inhibitor such as an anti-PD-1 antibody, anti-PDL-1 antibody, anti-CTLA4, etc., an immunosuppression antagonist, a pro-inflammatory agent/cytokine), therapeutic proteins, antibodies, or antigens (e.g., a tumor specific antigen).
  11. 11 . The RNA molecule of any one of claims 8 to 10, wherein the RNA molecule has a half-life that is more than that of a corresponding natural RNA molecule in a cellular environment.
  12. 12. The RNA molecule of any one of claims 8 to 11, having reduced formation of double stranded RNA compared to an RNA molecule with a 5' end not comprising the compound, stereoisomer, tautomer, or salt of any one of claims 1 to 7.
  13. 13. A drug product comprising the RNA molecule of any one of claims 8 to 12 and one or more pharmaceutically acceptable excipients.
  14. 14. A kit for capping an RNA molecule comprising the compound, stereoisomer, tautomer, or salt of any one of claims 1 to 7, and an RNA polymerase.
  15. 15. The kit of claim 14, wherein the RNA molecule is mRNA.
  16. 16. A method for making a capped RNA molecule from a polynucleotide template by in vitro transcription, comprising: (a) combining a plurality of nucleotides, the polynucleotide template and an RNA polymerase to produce a reaction mix; (b) incubating the reaction mix; and (c) adding the compound, stereoisomer, tautomer, or salt of any one of claims 1 to 7 to the mix in order to produce the capped RNA molecule.
  17. 17. The method of claim 16, wherein the RNA molecule is mRNA.
  18. 18. The method of claim 16 or claim 17, wherein at least a portion of the plurality of nucleotides are unmodified.
  19. 19. The method of any one of claims 16 to 18, wherein the at least a portion of the plurality of nucleotides are modified.
  20. 20. The method of any one of claims 16 to 19, wherein the polynucleotide template is a DNA template.

Description

ETHYL MODIFIED RNA CAPS AND METHODS OF USE BACKGROUND [0001] The present disclosure generally relates to RNA cap analogs, polynucleotides (e.g., mRNAs) containing the cap analogs, and methods of making and using the cap analogs. [0002] The naturally-occurring eukaryotic mRNA has a cap structure comprising an N7-methylated guanosine (m7G or 7mG) linked to the first nucleotide of the mRNA via a reverse 5' to 5' triphosphate linkage (5' ppp). The mRNA cap has an essential role in cap-dependent initiation of protein synthesis and functions as a protective group from 5' to 3' exonuclease cleavage and a unique identifier for recruiting protein factors for pre-mRNA splicing, polyadenylation and nuclear export. It also acts as the anchor for the recruitment of initiation factors that initiate protein synthesis and the 5' to 3' looping of mRNA during translation. [0003] Capping can be achieved on synthetically-derived mRNA using multiple strategies including post- transcriptional enzymatic capping and co-transcriptional capping. Several enzymatic steps are required for the conversion of the 5'-triphosphory I ated end of an mRNA transcript into a capped structure. The enzymes involved in these steps are referred to as "capping enzymes.” Co-transcriptional capping uses a "capping reagent” which can be incorporated into a growing mRNA chain during the transcriptional process without the need for additional enzymatic steps. The RNAs that result from these enzymatic steps are referred to as "5' capped RNAs” or simply "capped RNAs.” [0004] The mRNA cap structure is implicated in many aspects of mRNA efficacy, including translation efficacy, mRNA stability, and mRNA immunogenicity. However, little is known about the medicinal chemistry of how these cap structures bind to the relevant enzymes in either the capping process (i.e. during mRNA synthesis), or to the translation machinery enzymes such as el F4E. [0005] There remains a need for synthetic cap analogs that efficiently cap polynucleotides and provide polynucleotides with desired biological properties, such as capped RNAs with desirable translation efficiency. SUMMARY [0006] Provided herein are compounds of Formula (I), or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof: wherein X is O or S, R1 is OH or a polynucleotide, and R2 is H or Me. [0007] Also provided are RNA molecules, wherein the 5' end of the RNA molecule comprises a compound, stereoisomer, tautomer, or salt disclosed herein. Further provided are drug products comprising a capped RNA molecule disclosed herein and one or more pharmaceutically acceptable excipients. Further provided are kits for capping an RNA molecule comprising a compound disclosed herein, or stereoisomer, tautomer, or salt thereof, and an RNA polymerase. Further provided are methods for making a capped RNA molecule from a polynucleotide template by in vitro transcription, comprising: (a) combining a plurality of nucleotides, the polynucleotide template and an RNA polymerase to produce a reaction mix; (b) incubating the reaction mix; and (c) adding a compound disclosed herein, or a stereoisomer, tautomer, or salt thereof to the mix in order to produce the capped RNA molecule. BRIEF DESCRIPTION OF THE DRAWINGS [0008] FIG 1 A shows the "I VT”, or in vitro transcription yield for transcription of mRNAs containing RNA cap structures Cap 1, Compound 1 , or Compound 2. [0009] FIG 1 B shows the in vitro expression of mRNAs containing either RNA cap structure Cap 1 or RNA cap structure Compound 1 . [0010] FIG 1C shows the in vivo expression of mRNAs containing either RNA cap structure Cap 1 or RNA cap structure Compound 1 . [0011] FIG 1 D shows the levels of dsRNA produced during IVT reactions with mRNAs containing either RNA cap structure Cap 1 or RNA cap structure Compound 1 , as determined by sandwich ELISA. [0012] FIG 2A shows the "IVT”, or in vitro transcription yield for transcription of mRNAs containing RNA cap structures Cap 1, Cap 3, Cap 3, Compound 3, or Compound 4. [0013] FIG 2B shows the in vitro expression of mRNAs containing RNA cap structures Cap 1 , Cap 3, Cap 3, Compound 3, or Compound 4. [0014] FIG 2C shows the in vivo expression of mRNAs containing RNA cap structures Cap 1 , Cap 3, Cap 3, or Compound 3. [0015] FIG 2D shows the levels of dsRNA produced during IVT reactions with mRNAs containing RNA cap structures Cap 1, Cap 3, Cap 3, or Compound 3, as determined by sandwich ELISA. [0016] FIG 3A shows the expression of mRNAs containing no cap structures, or RNA cap structures Cap 1 or Compound 3. [0017] FIG 3B shows the degree of I FNy stimulation by mRNAs containing no cap structures, or RNA cap structures Cap 1 or Compound 3. [0018] FIG 3C shows the degree of I FNo stimulation by mRNAs containing no cap structures, or RNA cap structures Cap 1 or Compound 3. [0019] FIG 3D shows the degree of IP-10 stimulation by mRNAs containing no cap structures, or RNA cap structures Cap 1 or Compound 3. [0020] FIG 3E sho