Modified Replicable RNA CSE Evolution for Restored Replication
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Solution Overview
Problem
Modified nucleotides in self-amplifying RNA (saRNA) inhibit replication and translation, necessitating a need for saRNA molecules with sufficient replication and translation function despite containing modified nucleotides.
Innovation Solution
Identify sequence changes in replicable RNA molecules containing modified nucleotides to restore or improve their replication and translation capabilities through an in vitro evolution method, specifically targeting the 5′- or 3′-conserved sequence elements (CSE) to enhance interaction with replicase.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If modified nucleotides (e.g., N1-methyl-pseudouridine) are incorporated into replicable RNA to avoid innate immune response and improve translation, then immune compatibility and translation efficiency are improved, but replication ability is inhibited
Solution Approach 1:
The patent applies parameter changes by systematically varying nucleotide sequences in the 5′- and 3′-conserved sequence elements (CSEs) of replicable RNA molecules. Through in vitro evolution, specific sequence parameters were optimized to compensate for the inhibitory effect of modified nucleotides on replication, while maintaining the immune-compatible properties provided by the modifications.
Solution Approach 2:
The patent uses in vitro evolution to create multiple copies and variants of replicable RNA molecules with modified nucleotides. Through iterative replication and selection processes, optimal sequence copies were identified that restore replication function while preserving the benefits of nucleotide modification for immune compatibility.
2Productivity
If self-amplifying RNA (saRNA) is designed to replicate autonomously in cells to amplify vaccine antigen, then vaccine potency is improved, but innate immune response is strongly stimulated which inhibits effectiveness
Solution Approach 1:
The patent optimizes sequence parameters in the conserved sequence elements of saRNA molecules to achieve a balance between replication efficiency and immune compatibility. By modifying specific nucleotide sequences while maintaining the overall saRNA structure, the patent enables autonomous replication with reduced innate immune stimulation.
Solution Approach 2:
The patent uses modified nucleotides as intermediaries that mediate between the need for autonomous replication and the need to avoid strong innate immune responses. These modified nucleotides act as a bridge, allowing the saRNA to replicate effectively while being less recognizable to innate immune sensors.
3Reliability
If high amounts of saRNA (30 to 100 μg per dose) are administered to overcome innate immune inhibition and achieve sufficient immune response, then vaccine effectiveness is improved, but production time and cost increase significantly
Solution Approach 1:
The patent changes the sequence parameters of saRNA molecules to optimize the balance between replication efficiency and immune stimulation. This allows for reduced dosages while maintaining vaccine effectiveness, thereby reducing production time and costs associated with manufacturing and distributing large quantities of vaccine.
Data Source
AI summary
The present invention relates to methods for restoring or improving the ability of a modified nucleotide-containing replicable RNA to be replicated and/or translated. The method includes identifying nucleotide changes in the replicable RNA that compensate for the lowered ability of modified-nucleotide-containing replicable RNA molecules to replicate and/or be translated. The present invention also relates to modified nucleotide-containing replicable RNA molecules incorporating such identified nucleotide changes and the use of such replicable RNA molecules in therapy.