Stealth RNA Gene Expression System for Immune-Evasive Multi-Gene Delivery
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Solution Overview
Problem
Current gene introduction and expression techniques face challenges in efficiently introducing and persistently expressing multiple exogenous genes in animal cells, particularly human peripheral blood cells, while avoiding immune system activation and enabling easy gene removal, especially when the gene length exceeds 5,000 nucleotides or more.
Innovation Solution
Development of a stealth RNA gene expression system using negative-sense single-stranded RNA combined with optimized transcription start and termination signals, replication origins, and RNA-dependent RNA polymerase sequences, which are designed to be non-immunogenic, allowing for the simultaneous expression of up to ten genes without activating the innate immune system.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional gene introduction methods are used to efficiently introduce and express multiple exogenous genes, then gene expression efficiency is improved, but the innate immune system is activated and cytotoxicity increases
Solution Approach 1:
The patent changes the chemical structure parameters of the introduced genetic material by using chemically modified nucleosides and nucleotides (such as 5-methylcytidine, N6-methyladenosine, pseudouridine) instead of conventional unmodified nucleotides. This parameter change in molecular structure allows the RNA to evade immune recognition while maintaining gene expression functionality, thereby resolving the contradiction between expression efficiency and immune activation.
Solution Approach 2:
The patent employs transient RNA expression systems that do not require permanent integration into the host genome. The chemically modified RNA molecules serve as temporary, disposable carriers that deliver genetic information and are subsequently degraded by cellular mechanisms, avoiding long-term immune activation while achieving efficient transient gene expression.
2Adaptability or versatility
If multiple exogenous genes are introduced simultaneously to achieve complex gene expression, then gene functionality is improved, but vector size and complexity increase
Solution Approach 1:
The patent merges multiple exogenous genes into a single chemically modified RNA transcript using polycistronic design or IRES (Internal Ribosome Entry Site) elements. This combining approach allows simultaneous introduction and expression of multiple genes (such as OCT4, SOX2, KLF4, c-MYC for iPS cell generation) through one unified vector, reducing overall system complexity while maintaining versatile gene expression capability.
Solution Approach 2:
The chemically modified RNA vector system serves multiple functions: it acts as a delivery vehicle, a template for protein synthesis, and a regulator of gene expression levels. The universal applicability of the chemically modified nucleotide platform across different gene combinations and cell types enables a single system design to handle diverse gene introduction requirements without increasing complexity.
3Adaptability or versatility
If gene introduction is performed in cells with low proliferative ability, then applicability to clinical applications is improved, but gene expression efficiency decreases
Solution Approach 1:
The patent replaces conventional DNA-based gene delivery mechanisms that rely on cellular proliferation and nuclear integration with RNA-based delivery that functions directly in the cytoplasm. The chemically modified RNA molecules can be translated by ribosomes without requiring nuclear entry or cell division, enabling efficient gene expression in post-mitotic cells such as neurons, cardiomyocytes, and peripheral blood cells.
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
This approach enables efficient and persistent gene expression in various tissue cells, including those with low proliferative ability, with low cytotoxicity and interferon induction, facilitating regenerative medicine and biopharmaceutical production by avoiding immune recognition and allowing for easy gene removal.
Implementation Method 1
negative-sense single-stranded RNA combined with optimized transcription start and termination signals, replication origins, and RNA-dependent RNA polymerase sequences
Data Source
AI summary
The present invention enables simultaneous and stable expression of a plurality of foreign genes by using a stealthy RNA gene expression system that is a complex that does not activate the innate immune mechanism and is formed from an RNA-dependent RNA polymerase, a single-strand RNA binding protein, and negative-sense single-strand RNAs including the following (1) to (8): (1) a target RNA sequence that codes for any protein or functional RNA; (2) an RNA sequence forming a noncoding region and derived from mRNA expressed in animal cells; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA polymerase; (4) a transcription termination signal sequence recognized by the polymerase; (5) an RNA sequence containing a replication origin recognized by the polymerase; (6) an RNA sequence that codes for the polymerase and of which codons are optimized for the species from which an introduction target cell is derived; (7) an RNA sequence that codes for a protein for regulating the activity of the polymerase and of which codons are optimized for the species from which the introduction target cell is derived; and (8) an RNA sequence that codes for the single-strand RNA binding protein and of which codons are optimized for the species from which the introduction target cell is derived.