IP Library › Granted Patent US 12,600,998
Granted Patent B2
US 12,600,998 · App. 17/502,816 · Granted Apr 14, 2026

Large scale synthesis of messenger RNA

Inventors: Jonathan Abysalh (Lexington, MA); Anusha Dias (Lexington, MA); Jorel E. Vargas (Lexington, MA); Dustin Cooper (Lexington, MA); Frank DeRosa (Lexington, MA)
Assignee: TRANSLATE BIO, INC.
C12P19/34A61K31/713
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,600,998
App. No.
17/502,816
Granted
Apr 14, 2026
Kind
B2
Abstract

The present invention provides, among other things, methods for large-scale production of a composition comprising full-length messenger RNA product that is substantially free of double-stranded RNA, and compositions produced using such methods and uses thereof. The present invention is based, in part, on the surprising discovery that mRNA product produced by in vitro transcription using an SP6 RNA polymerase is substantially free of double-stranded RNA. In one aspect, the present invention provides methods of generating large-scale mRNA product for mRNA therapy without need for a chromatography step.

Claims (19)

1 . A method of production of a composition comprising full-length messenger RNA (mRNA), comprising synthesizing in vitro mRNA from a DNA template using an SP6 RNA polymerase, wherein the mRNA is at least 500 bases in length, wherein the weight ratio of the DNA template to the SP6 RNA polymerase is between 1:1.5 and 1:2.5, wherein at least 100 mg of mRNA is synthesized in a single batch, and wherein the composition contains less than 1% of double-stranded RNA (dsRNA) by weight.

2 . The method of claim 1 , wherein the mRNA is synthesized at a non-denaturing condition.

3 . The method of claim 1 , wherein the in vitro synthesis of mRNA is performed in a buffer comprising 25 mM Tris HCl, 2 mM spermidine, 25 mM MgCl 2 , 0.5 mM NaCl, and pH 7.5.

4 . The method of claim 1 , wherein the method further comprises a step of capping and/or tailing the synthesized mRNA.

5 . The method of claim 1 , wherein the method does not include a step of specifically removing the dsRNA.

6 . The method of claim 1 , wherein the SP6 RNA polymerase is a recombinant SP6 RNA polymerase.

7 . The method of claim 1 , wherein the DNA template comprises an SP6 promoter operably linked to a DNA sequence encoding the mRNA sequence to be synthesized.

8 . The method of claim 7 , wherein the DNA template sequence is optimized to reduce the chance of a hairpin structure forming in the synthesized mRNA.

9 . The method of claim 1 , wherein the mRNA is synthesized in a reaction mixture comprising NTPs at a concentration ranging from 1-10 mM for each NTP.

10 . The method of claim 1 , wherein the mRNA is synthesized at a temperature ranging from 37-42° C.

11 . The method of claim 9 , wherein the NTPs comprise modified NTPs.

12 . The method of claim 1 , wherein the weight ratio of the DNA template to the SP6 RNA polymerase is 1:1.5.

13 . The method of claim 1 , wherein the weight ratio of the DNA template to the SP6 RNA polymerase is about 1:2.

14 . The method of claim 1 , wherein the weight ratio of the DNA template to the SP6 RNA polymerase is 1:2.5.

15 . The method of claim 11 , wherein the modified NTPs comprise a modified uridine.

16 . The method of claim 15 , wherein the modified uridine is pseudouridine.

17 . The method of claim 16 , wherein the pseudouridine is N-1-methyl-pseudouridine.

18 . The method of claim 1 , wherein the DNA template is plasmid DNA.

19 . The method of claim 1 , wherein at least 10 mg of the DNA template is used to produce at least 1 gram of mRNA.

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 11, 2024
From: ABYSALH, JONATHAN; DIAS, ANUSHA; VARGAS, JOREL E.; COOPER, DUSTIN; DEROSA, FRANK
To: TRANSLATE BIO MA, INC.
Reel/Frame 066713/0864 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 23, 2022
From: ABYSALH, JONATHAN; DIAS, ANUSHA; VARGAS, JOREL E.; COOPER, DUSTIN
To: TRANSLATE BIO MA, INC.
Reel/Frame 059980/0879 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 23, 2022
From: TRANSLATE BIO MA, INC.
To: TRANSLATE BIO, INC.
Reel/Frame 059981/0325 →
Continuity (2)
Provisional Application 63092274 · Oct 15, 2020
Related Publication 20220136022A1 · May 5, 2022
References Cited (37)
US 11253605B2 · Dias · 2022 [cited by examiner]
US 11560562B2 · Dias · 2023 [cited by examiner]
US 20160326575A1 · Von Der Mulbe et al. · 2016 [cited by applicant]
US 20180161451A1 · Fotin-Mleczek et al. · 2018 [cited by applicant]
US 20180258423A1 · Dias et al. · 2018 [cited by applicant]
CN 111212905A · 2020 [cited by applicant]
TW 202023580A · 2020 [cited by applicant]
WO WO2012170930A1 · 2012 [cited by applicant]
WO WO2015188933A1 · 2015 [cited by applicant]
WO WO2016149508A1 · 2016 [cited by applicant]
WO WO2016180430A1 · 2016 [cited by applicant]
WO WO2018053209A1 · 2018 [cited by applicant]
WO WO2018157153A1 · 2018 [cited by applicant]
WO WO2018157154A2 · 2018 [cited by applicant]
WO WO2019036682A1 · 2019 [cited by applicant]
WO WO2019207060A1 · 2019 [cited by applicant]
WO WO2020023888A2 · 2020 [cited by applicant]
“Between” definition provided by Merriam Webster's Dictionary, downloaded from https://www.merriam-webster.com/dictionary on Nov. 4, 2023. [cited by examiner]
“About” definition provided by Merriam Webster's Dictionary, downloaded from https://www.merriam-webster.com/dictionary on Nov. 4, 2023. [cited by examiner]
Stump et al. (Nucleic Acids Research, 1993 vol. 21:5480-5484). [cited by examiner]
Alton, E. et al., “A randomised, double-blind, placebo-controlled trial of repeated nebulisation of non-viral cystic fibrosis transmembrane conductance regulator (CFTR) gene therapy in patients with cystic fibrosis,” Ef… [cited by applicant]
Anonymous, “RiboMAX™ Large Scale RNA Production Systems-SP6 and T7”, Promega, pp. 1-14 (2017). [cited by applicant]
Brito et al., “Self-Amplifying mRNA Vaccines”, Advances in Genetics, 89: 179-233 (2015). [cited by applicant]
Gonzalez-Perez et al., “Scaling up in vitro transcription synthesis of RNA standards for competitive quantitative RT-PCR: Looking for bigger yields”, Anal Biochem., 385(1): 179-81 (2009). [cited by applicant]
Gurevich et al., “Preparative in vitro mRNA Synthesis Using SP6 and T7 RNA Polymerases”, Analytical Biochemistry, 195: 207-213 (1991). [cited by applicant]
He et al., “Rapid Mutagensis and Purification of Phage RNA Polymerases”, Protein Expression and Purification, 9: 142-151 (1997). [cited by applicant]
International Preliminary Report on Patentability for PCT/US2018/020008, 7 pages, (dated Sep. 6, 2019). [cited by applicant]
International Search Report and Written Opinion for PCT/US2018/020008, 18 pages, (dated May 11, 2018). [cited by applicant]
Krieg et al., “In Vitro RNA Synthesis with SP6 RNA Polymerase”, Methods in Enzymology, 155: 397-415 (1987). [cited by applicant]
Lee et al., “Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator”, Nucleic Acids Research, vol. 38, No. 18, pp. 6045-6053 (2010). [cited by applicant]
Pascolo, S., “Vaccination with messenger RNA”, Methods in Molecular Medicine; 127: 23-40 (2006). [cited by applicant]
Robinson et al., “Lipid Nanoparticle-Delivered Chemically Modified mRNA Restores Chloride Secretion in Cystic Fibrosis”, Molecular Therapy, 26(8): 1-13 (2018). [cited by applicant]
Skeidsvoll et al., “Analysis of RNA by capillary electrophoresis”, Electrophoresis, 17: 1512-1517 (1996). [cited by applicant]
Thurston et al.,“Electrophoresis of RNA Denatured with Glyoxal or Formaldehyde”, Methods in Molecular Biology, 4: 1-11 (1988). [cited by applicant]
Yang et al., “Determination of RNA degradation by capillary electrophoresis with cyan light-emitted diode-induced fluorescence”, Journal of Chromatography A 2012; 1239: 78-84 (2012). [cited by applicant]
Yoshioka et al., “Efficient generation of human iPSCs by a synthetic self-replicative RNA”, Cell Stem Cell, 13(2): 246-54 (2013). [cited by applicant]
International Search Report and Written Opinion for PCT/US2021/055221 dated Feb. 3, 2022 (12 pages). [cited by applicant]