Self-Replicating RNA Replicons for High Expression, Low Immunogenicity
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Solution Overview
Problem
Current self-replicating RNA molecules, such as saRNA, face challenges in achieving high and sustained protein expression while minimizing immune response and maintaining low dosage requirements, with existing sequences like VEEV TC83 replicons falling short in clinical applications.
Innovation Solution
Development of replicable RNA molecules utilizing nonstructural proteins from specific viruses, such as Mosso das Pedras virus, with optimized UTRs and cis expression of accessory proteins, enhancing protein expression levels and reducing immunogenicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If self-replicating RNA molecules are used to achieve sustained protein expression, then protein expression duration is improved, but immune response intensity increases
Solution Approach 1:
The patent applies local quality by optimizing specific regions of the RNA molecule (5' UTR and 3' UTR) with sequences derived from VEEV TC83 replicon, while keeping other regions modified or replaced. This localized optimization allows the RNA to maintain replicability and reduce immunogenicity without compromising the overall self-replicating function, thereby achieving sustained expression with reduced immune response.
Solution Approach 2:
The patent employs parameter changes by modifying the sequence identity of the RNA molecule to be at least 85%, 90%, 95%, or 99% identical to the VEEV TC83 replicon sequence. By adjusting this sequence identity parameter, the patent optimizes the balance between replicability and immunogenicity, achieving sustained protein expression while minimizing immune recognition.
2Productivity
If high protein expression is achieved through self-replicating RNA, then productivity is improved, but RNA dosage requirements increase
Solution Approach 1:
The patent applies self-service by designing an RNA molecule that contains its own replication machinery through the incorporation of VEEV TC83 replicon sequences. The RNA molecule can autonomously replicate within host cells without requiring continuous external supplementation, enabling high protein expression levels from low initial dosages and reducing the need for repeated administrations.
3Reliability
If existing saRNA sequences like VEEV TC83 are used, then replicability is achieved, but protein expression levels are insufficient
Solution Approach 1:
The patent applies composite materials by creating a hybrid RNA structure that combines elements from different sources: the VEEV TC83 replicon sequences for replicability, optimized 5' and 3' UTR regions for enhanced expression, and customizable coding regions for specific protein products. This composite structure integrates the strengths of different RNA components to achieve both high replicability and high protein expression levels.
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 new RNA molecules achieve significantly higher protein expression and lower immunogenicity, addressing the limitations of existing saRNA sequences by leveraging optimized viral replicons and UTRs.
Implementation Method 1
the saRNA comprises a very large open reading frame at the 5′ end encoding 4 nonstructural proteins (nsP) of the positive-strand RNA virus... which assemble to form an RNA-dependent RNA polymerase complex, also known as an RNA replicase. The RNA polymerase complex first synthesizes a complementary antisense strand RNA from the sense strand RNA.
Data Source
AI summary
The present application relates to a replicable RNA molecule and the use thereof. The replicable RNA molecule comprises, from the 5′ end to the 3′ end, a 5′ cap, a 5′ UTR, an open reading frame encoding an RNA replicase, a promoter, a sequence of interest, a 3′ UTR and a poly(A) tail, wherein the RNA replicase is capable of amplifying the replicable RNA molecule and is capable of amplifying an RNA molecule containing the sequence of interest and 3′ UTR, wherein the RNA replicase is a nonstructural protein or a functional variant thereof derived from Mosso das Pedras virus (MDPV), Everglades virus (EVEV), Rio Negro virus (RNV), Mucambo virus (MUCV), Highlands J virus (HJV), Pixuna virus (PIXV), Trocara virus (TROV), Cabassou virus (CABV), Tonate virus (TONV), Bebaru virus (BEBV), Fort Morgan virus (FMV), Getah virus (GETV) or Ndumu virus (NDUV).