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EP-4735628-A1 - METHODS OF ANALYZING AND CHARACTERIZING MESSENGER RIBONUCLEIC ACID (MRNA) AND POLYADENYLATION ON MRNA

EP4735628A1EP 4735628 A1EP4735628 A1EP 4735628A1EP-4735628-A1

Abstract

The inventions provide methods for characterizing messenger RNAs (mRNA) and polyadenylation on mRNAs. The disclosed inventions are developed for characterizing mRNA and poly(A) tail length on mRNA samples. These inventions can be used in release testing of mRNA drug substrate (DS) or drug product (DP), and can provide direct read-out of mRNA integrity and poly(A) tail length. The inventions further provide methods of analyzing the condition of mRNAs in samples. Characterized mRNA samples obtained by all of the inventive methods also are part of the inventions disclosed herein.

Inventors

  • DI GRANDI, Deanna
  • FU, YUE
  • MUTHUSAMY, Kathir
  • PALACKAL, NISHA
  • DAYEH, Daniel

Assignees

  • Regeneron Pharmaceuticals, Inc.

Dates

Publication Date
20260506
Application Date
20240701

Claims (20)

  1. 1. A method for characterizing a polyadenylated region on a messenger ribonucleic acid (mRNA) that further comprises a coding region, wherein the method comprises the steps of: (a) adjusting mRNA concentration in an mRNA sample based upon mRNA length; (b) treating the mRNA sample with RNase T1 to remove the coding region and allow the polyadenylated region to remain; (c) incubating the RNase T1 treated sample with oligo-dT beads; (d) eluting the polyadenylated region bound to the oligo-dT beads; (e) removing residual salts from the elution of step (d); and (f) analyzing the polyadenylated region of step (e) using capillary gel electrophoresis (CGE).
  2. 2. The method according to claim 1, wherein step (d) uses nuclease free water.
  3. 3. The method according to claims 1-2, wherein step (e) uses a Zeba spin, dialysis, tangential flow flotation, or CAT cartridges.
  4. 4. The method according to claims 1-3, wherein step (f) uses a binding buffer comprising 20 mM Tris-HCl, a pH of 7.5, 2 mM EDTA, and 1000 mM LiCl.
  5. 5. The method according to claims 1-4, wherein step (f) uses a separation buffer comprising Tris-Borate-Urea.
  6. 6. The method according to claims 1-5, wherein step (f) uses a wash buffer comprising 10 mM Tris-HCl, a pH of 7.5, 1 mM EDTA, and 150 mM LiCl.
  7. 7. The method according to claims 1-6, further comprising, prior to step (f), mixing the mRNA sample with a population of mRNA ladders, wherein each mRNA ladder comprises at least one poly(A) sequence member having a guanine nucleotide spacer, wherein the mRNA ladder can have a nucleotide length of 12 to 250 nucleotides.
  8. 8. The method according to claim 7, wherein the mRNA ladder can have a nucleotide length of 20 to 130 nucleotides.
  9. 9. The method according to claims 1-8, wherein the RNase T1 sample comprises about 0.5 M salt to allow binding to the oligo-dT beads.
  10. 10. The method according to claims 1-9, wherein step (e) facilitates CGE.
  11. 11. The method according to claims 1-10, where step (f) utilizes an ultraviolet (UV) detector for detecting the poly adenylated region.
  12. 12. The method according to claim 11, wherein the UV detector uses light at a wavelength of 250 to 260 nm.
  13. 13. The method according to claim 12, wherein the UV detector uses light at a wavelength of 254 nm.
  14. 14. The method according to claim 7, wherein an mRNA ladder comprises a poly(A) sequence member having 10 to 25 adenine residues with a guanine nucleotide spacer, and wherein the mRNA ladder has a hydroxyl group at the 3’ end.
  15. 15. The method according to claim 14, wherein the mRNA ladder further comprises a plurality of poly(A) sequence members having 10 to 25 adenine residues with a guanine nucleotide spacer.
  16. 16. The method according to claim 7, wherein the population of mRNA ladders comprises (i) a first mRNA ladder comprising at least one poly(A) sequence member having 10 to 25 adenine residues with a guanine nucleotide spacer, and wherein the first mRNA ladder has a hydroxyl group at the 3’ end, and (ii) a second mRNA ladder comprising at least one poly(A) sequence member having 10 to 25 adenine residues with a guanine nucleotide spacer, and wherein the second mRNA ladder has a phosphate group at the 3’ end.
  17. 17. The method according to claim 16, wherein the first mRNA ladder and the second mRNA ladder each further comprise at least one Nl-methyl-pseudouridine nucleotide.
  18. 18. A characterized sample of a polyadenylated region of a messenger RNA (mRNA) that further comprises a coding region, wherein the characterized sample is produced according to the steps of: a) adjusting mRNA concentration in an mRNA sample based upon mRNA length; (b) treating the mRNA sample with RNase T1 to remove the coding region and allow the polyadenylated region to remain; (c) incubating the RNase T1 treated sample with oligo-dT beads; (d) eluting the polyadenylated region bound to the oligo-dT beads; (e) removing residual salts from the elution of step (d); and (f) analyzing the polyadenylated region of step (e) using capillary gel electrophoresis (CGE).
  19. 19. The characterized sample according to claim 18, wherein step (d) uses nuclease free water.
  20. 20. The characterized sample according to claims 18-19, wherein step (e) uses a Zeba spin, dialysis, tangential flow flotation, or CAT cartridges.

Description

METHODS OF ANALYZING AND CHARACTERIZING MESSENGER RIBONUCLEIC ACID (mRNA) AND POLYADENYLATION ON mRNA CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63/524,379 filed on June 30, 2023, and U.S. Provisional Application No. 63/528,721 filed on July 25, 2023. The above-referenced applications are incorporated herein by reference in their entirety. REFERENCE TO ELECTRONIC SEQUENCE LISTING [0002] The application contains a sequence listing which has been submitted electronically in .xml format and is hereby incorporated by reference in its entirety. The .xml file, created on June 26, 2024, is named “REGN 11474. xml” and is 75,949 bytes in size. The sequence listing contained in this .xml file is part of the specification and is hereby incorporated by reference herein in its entirety. TECHNICAL FIELD OF THE INVENTIONS [0003] The present inventions provide for improved methods of analyzing and characterizing messenger ribonucleic acid (mRNA) and polyadenylation on mRNA. The inventions provide, among other things, improved approaches for evaluating, designing, producing and utilizing mRNA in the production of protein and other uses. Messenger RNA preparations provided by the inventive methods also are part of the inventions. BACKGROUND OF THE INVENTIONS [0004] Messenger RNA has in the past been considered for medical therapies. Since the onset of the COVID- 19 pandemic, in vitro transcribed (IVT) messenger ribonucleic acid (mRNA) has gained unprecedented attention and quickly proven itself to have the potential to become a new class of therapeutics for drug and vaccine development. Advances in addressing the challenges of IVT mRNA, particularly related to controlling translational efficacy and immunogenicity, will be needed for the successful translation of mRNA-based therapeutics from ideas to clinical approval. [0005] Messenger RNA is normally considered to be the information shuttle between deoxyribonucleic acid (DNA) and translated proteins. Several components of mRNA are essential to its function, stability and translation efficiency, including a cap structure at a 5’ end, a 5’ untranslated region (UTR) and a 3’ UTR, and a polyadenylation (poly(A)) tail at a 3’ end. Notably, the presence of the 5’ cap and 3’ poly(A) tail are necessary for translation initiation and protein production; which generally occurs when the poly(A) tail interacts with a poly(A) binding protein, the 5’ cap and eukaryotic initiation factors (elF). [0006] Immediately after a gene in a eukaryotic cell is transcribed, the new mRNA molecule undergoes several modifications, which is known as mRNA processing. These modifications alter both ends of the primary mRNA transcript to produce a mature mRNA molecule. The processing of the 3' end adds a poly(A) tail to the mRNA molecule. First, the 3' end of the transcript is cleaved to free a 3' hydroxyl. Then an enzyme called poly-A polymerase adds a chain of adenine nucleotides to the mRNA. This process, called polyadenylation, adds a poly(A) tail that is approximately 100 to 250 nucleotides (nt) in length. The poly(A) tail makes the RNA molecule more stable and prevents its degradation. Additionally, the poly(A) tail allows the mature messenger RNA molecule to be exported from the nucleus and translated into a protein by ribosomes in the cytoplasm. The presence of a poly(A) tail is a critical quality attribute for a messenger RNA vaccine. Messenger RNA molecules with and without a poly(A) tail (that is, tailless mRNA) can be separated and quantitated using ion pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC, IP-RP-LC or IPRP-LC). [0007] To mimic mRNA produced in cells, medicinal mRNA molecules experience deadenylation and terminate with a stretch of adenosines at their 3’ end and have various lengths. This deadenylation is the first step in mRNA decay as shorter poly(A) tails normally lead to a shorter half-life of mRNA molecules. The shortening of the 3'-poly(A) tail is the ratelimiting step in the degradation of most mRNAs and is important toward understanding the length impact of poly(A) tail. [0008] For mRNA therapeutics or vaccines, the addition of poly(A) tails can be accomplished in three ways, including encoding poly(A) signal (PAS) into a template plasmid DNA, adding a poly(A) sequence to polymerase chain reaction (PCR) primers, or by adding a poly(A) sequence post IVT using polyadenylase. While each approach has both advantages and disadvantages, encoding PAS into a template plasmid DNA or adding a poly(A) sequence to PCR primers are of the most popular techniques used for mRNA-based therapeutics or vaccines. [0009] Messenger RNA can degrade both in the coding regions and the poly(A) tail. Methods of analyzing and characterizing the entire mRNA and the poly(A) tail are needed. [0010] To characterize the length and distribution of poly(A) tail in mRNA, current United States P