In vitro transcription technologies
Inventors
Rabe, Irena • Buff, Maximilian • Ziegenhals, Thomas • Drögemüller, Johanna • Kuhn, Andreas • Fesser, Stephanie • Combs, Rodney Gene • Eschmann, Nicole Star • Romine, Jennifer Ann Schoborg • Williamson, Jenna Kathryn
Assignees
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Publication Number
US-12305210-B2
Publication Date
2025-05-20
Expiration Date
Abstract
The present disclosure provides technologies for in vitro transcription reactions, particularly for production of pharmaceutical grade RNA, and in some embodiments for large scale production.
Core Innovation
The invention provides an in vitro transcription method for producing an RNA molecule by creating a reaction mixture under reaction conditions to form the RNA molecule. The reaction mixture comprises a nucleic acid polymerase and a nucleic acid template, and the nucleotide composition is defined by molar ratios of total CTP and/or one or more CTP analog(s) relative to total GTP and/or one or more GTP analog(s), total UTP and/or one or more UTP analog(s), and total ATP and/or one or more ATP analog(s).
At least one of the molar ratios deviates from an equal 1:1:1:1 CTP:GTP:UTP:ATP balance, with minimum ratios including a at least 1.25, b at least 1.25, and/or c at least 1.10. The method is independent of the sequence of the RNA molecule, and the disclosed embodiments further define reaction mixture components including a reaction buffer, an RNase inhibitor, inorganic pyrophosphatase, salts, a reducing agent, and spermidine.
The described approach addresses quality and performance during IVT of therapeutic RNA, including mRNA, by improving RNA integrity and RNA capping level/efficiency while reducing residual double-stranded RNA (dsRNA). The disclosure also includes quality characterization endpoints including RNA integrity, RNA concentration, residual dsRNA, and capping, and relates to 5′ cap structures, including Cap1/Cap2 cap structures, cap-proximal nucleotides, and cap analog structures such as ARCA and related anti-reverse cap analog configurations.
Claims Coverage
The claim coverage centers on sequence-independent in vitro transcription nucleotide-ratio control with three inventive features directed to minimum molar ratios of total CTP relative to total GTP, UTP, and ATP, together with dependent refinements for reaction formulation, nucleotide addition timing, and quality outcomes.
Sequence-independent nucleotide molar-ratio control in IVT
An in vitro transcription method where the reaction mixture comprises a nucleic acid polymerase and a nucleic acid template, and the nucleotide feed is defined by molar ratios a, b, and/or c of total CTP and/or CTP analog(s) to total GTP and/or GTP analog(s), to total UTP and/or UTP analog(s), and/or to total ATP and/or ATP analog(s), respectively, with a at least 1.25 and/or b at least 1.25 and/or c at least 1.10, wherein the molar ratio of total CTP to GTP to UTP to ATP is not 1:1:1:1 and the method is independent of the sequence of the RNA molecule.
Residual dsRNA level constraints
The method results in residual dsRNA during and/or after transcription at least about 25–300 pg dsRNA per µg RNA, inclusive of the listed thresholds.
RNA integrity improvement relative to nonconforming ratios
The method increases RNA integrity of the produced RNA molecules by at least about 1%–20% compared with in vitro transcription using a reaction mixture where a is not at least 1.25, b is not at least 1.25, and/or c is not at least 1.10.
Expanded reaction mixture formulation components
The method uses a reaction mixture additionally containing one or more of a reaction buffer, an RNase inhibitor, a pyrophosphatase, one or more salts, a reducing agent, and spermidine.
Timed nucleotide addition before and after transcription start
Parts of the total CTP, GTP, UTP, and/or ATP and/or their analogs are added before and/or at the start of transcription, with remaining parts added after transcription begins.
Therapeutic RNA production
The method includes producing the RNA as a therapeutic RNA.
Overall, the claims focus on an IVT method that departs from a 1:1:1:1 CTP:GTP:UTP:ATP nucleotide ratio by enforcing minimum molar-ratio thresholds while remaining independent of RNA sequence. Dependent coverage further narrows the method with residual dsRNA and RNA integrity constraints and adds defined reaction components, nucleotide addition timing, and therapeutic RNA production.
Stated Advantages
Reduces residual double-stranded RNA (dsRNA).
Improves RNA integrity.
Improves capping level/efficiency.
Enables therapeutic/pharmaceutical-grade RNA use cases.
Documented Applications
Production of therapeutic RNA, including therapeutic mRNA, via in vitro transcription (IVT) at large scale and in vitro.
Use in mRNA vaccine contexts, including mRNA vaccines (BNT162 constructs).
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