In Vitro RNA Synthesis With Osmolytes for Low-Immunogenicity Yield
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Solution Overview
Problem
Existing RNA therapies face challenges in reducing the innate immune response to exogenous RNA, particularly due to contaminants produced during in vitro transcription, which can lead to decreased efficacy and high production costs.
Innovation Solution
Incorporating osmolytes, such as betaine, into in vitro transcription reactions allows for high yield synthesis of RNA at elevated temperatures using wild-type T7 RNA polymerase, reducing immunogenicity and cellular toxicity while maintaining high expression levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If conventional in vitro transcription methods are used at standard temperatures, then RNA synthesis can proceed, but double-stranded RNA by-products are formed which trigger immune pathways and decrease therapeutic efficacy
Solution Approach 1:
The patent applies parameter changes by elevating the transcription temperature from standard temperatures (e.g., 37°C) to elevated temperatures (e.g., 45-60°C). This temperature parameter change stabilizes the RNA polymerase and reduces the formation of double-stranded RNA by-products, thereby decreasing immunogenicity while maintaining high RNA yields
Solution Approach 2:
The patent uses an osmolyte (such as betaine, glycerol, or other compatible solutes) as an intermediary substance that facilitates transcription at elevated temperatures. The osmolyte acts as a mediator that stabilizes the RNA polymerase structure and enhances transcription efficiency at temperatures where the polymerase would normally be unstable, enabling reduced immunogenicity without sacrificing productivity
2Stability of the object's composition
If wild-type bacteriophage RNA polymerases are used at elevated temperatures without osmolytes, then transcription stability improves, but the polymerase does not function normally at such temperatures
Solution Approach 1:
The osmolyte serves as an intermediary that enables wild-type bacteriophage RNA polymerases to function reliably at elevated temperatures. The osmolyte stabilizes the polymerase structure and maintains its catalytic activity, effectively bridging the gap between thermal stability and functional reliability for polymerases that would normally be inactive at these temperatures
Solution Approach 2:
By changing the temperature parameter to elevated levels (45-60°C) and combining this with osmolyte addition, the patent creates conditions where wild-type polymerases achieve both stability and functional reliability. The parameter change triggers structural adjustments in the polymerase that, when combined with osmolyte stabilization, result in reliable function at temperatures where it would normally fail
3Object-affected harmful factors
If chemically modified nucleotides are used to reduce immunogenicity, then immune response decreases, but costly purification processes and licensing issues arise
Solution Approach 1:
The patent employs a simple, inexpensive osmolyte additive (such as betaine or glycerol) instead of complex chemically modified nucleotides. This cheap, readily available substance achieves the same immunogenicity reduction without requiring expensive purification processes or specialized licensing, simplifying manufacturing while maintaining effectiveness
Solution Approach 2:
The patent extracts and eliminates the need for chemically modified nucleotides and their associated complex purification and licensing requirements. By using conventional nucleotides with an osmolyte additive, the method removes the manufacturing complexity while achieving the same therapeutic outcome of reduced immune response
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method produces RNA with reduced immunostimulatory properties and lower double-strand RNA levels, enhancing therapeutic potency and viability of host cells, while maintaining high yield and efficiency.
Implementation Method 1
one or more osmolytes can be used as a means to facilitate in vitro transcription (e.g., in vitro RNA transcription) at an elevated temperature with an RNA polymerase (e.g., a wild-type bacteriophage RNA polymerases) that do not normally function at such an elevated temperature in the absence of an osmolyte
Implementation Method 2
incubating an in vitro transcription mixture, thereby producing an RNA product that comprises a plurality of single-stranded RNA molecules
Implementation Method 3
at least one RNA polymerase that recognizes the RNA polymerase promoter sequence
Data Source
AI summary
Compositions and methods for synthesizing an RNA product are provided herein. For example, the present disclosure provides a method of producing an RNA product comprising incubating an in vitro transcription mixture, thereby producing an RNA product that comprises a plurality of single-stranded RNA molecules. In some embodiments, an in vitro transcription mixture comprises a DNA template comprising an RNA polymerase promoter sequence operatively linked to a target sequence; at least one RNA polymerase that recognizes the RNA polymerase promoter sequence; a plurality of ribonucleotides comprising at least two different types of ribonucleotides, each type comprising a different nucleoside; and a transcription buffer comprising an osmolyte.