Circular RNA Reprogramming Factors for iPSC Generation

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Solution Overview

Problem

Current methods for producing induced pluripotent stem cells (iPSCs) using reprogramming factors like Oct3/4, Klf4, Sox2, Nanog, Lin28, and c-Myc face challenges such as genome integration risks, laborious processes, and immunogenicity issues with self-replicating RNA systems and mRNA-based approaches.

Innovation Solution

The use of circular RNAs (circRNAs) encoding these reprogramming factors, which can be combined with lipid nanoparticles (LNPs) to generate integration-free iPSCs, offering improved stability and reduced immunogenicity, and potentially increasing reprogramming efficiency and duration of protein expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If self-replicating RNA systems are used for reprogramming, then reprogramming efficiency is improved, but the risk of genome integration increases

Engineering Contradiction:

Improvereprogramming efficiencyVSAvoidgenome integration risk

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent extracts the reprogramming function from self-replicating RNA systems and transfers it to circular RNA molecules that encode reprogramming factors. These circular RNAs are delivered via lipid nanoparticles, separating the reprogramming function from the problematic self-replication mechanism while maintaining high reprogramming efficiency without genome integration risk.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent introduces circular RNA molecules as intermediary carriers that encode reprogramming factors. These circular RNAs serve as a safe intermediary that delivers the reprogramming function without the harmful self-replication and genome integration properties of traditional RNA systems, using lipid nanoparticles as delivery vehicles.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If mRNA-based reprogramming is used, then reprogramming capability is achieved, but the process becomes laborious and requires multiple transfections

Engineering Contradiction:

Improvereprogramming capabilityVSAvoidprocess complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent segments the reprogramming function into circular RNA molecules that are pre-assembled with lipid nanoparticles. This segmentation allows the reprogramming factors to be delivered in a single, integrated unit rather than requiring multiple separate transfections of mRNA, simplifying the overall process while maintaining reprogramming capability.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent performs preliminary action by pre-assembling the circular RNA molecules with lipid nanoparticles before delivery. This pre-preparation eliminates the need for multiple transfection steps required by mRNA-based methods, as the circular RNA-LNP complex is designed to deliver all necessary reprogramming factors in a single administration.

Inventive Principle:

Principle #10Preliminary action

3Productivity

If exogenous mRNA is used for reprogramming, then reprogramming is achieved, but immunogenicity increases requiring immune evasion factors

Engineering Contradiction:

Improvereprogramming achievementVSAvoidimmunogenicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent changes the fundamental parameter of the RNA structure from linear mRNA to circular RNA. This structural parameter change fundamentally alters the immunogenicity profile, as circular RNAs do not trigger the same immune responses as exogenous mRNA. The circular structure prevents recognition by immune sensors while maintaining the ability to encode and express reprogramming factors.

Inventive Principle:

Principle #35Parameter changes

4Reliability

If circular RNAs encoding reprogramming factors are used, then genome integration risk is reduced, but the complexity of RNA synthesis increases

Engineering Contradiction:

Improvegenome integration safetyVSAvoidRNA synthesis complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent replaces complex enzymatic RNA synthesis mechanisms with in vitro transcription using T7 RNA polymerase. This substitution simplifies the synthesis process by using a well-characterized, high-yield bacterial polymerase that can efficiently produce circular RNAs with high purity, eliminating the need for more complex eukaryotic transcription systems while ensuring genome integration safety through the circular structure.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Data Source

PatentUS20230332182A1Compositions and methods for cellular reprogramming using circular RNA

Publication Date: 2023.10.19 ELEVATEBIO TECHNOLOGIES INC

AI summary

Provided herein are recombinant circular RNAs comprising at least one protein-coding nucleic acid sequence, wherein the protein-coding nucleic acid sequence encodes a reprogramming factor (e.g., a transcription factor), wherein the reprogramming factor is Oct3/4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc, or a fragment or variant thereof. Also provided herein are methods of producing induced pluripotent stem cells (iPSC), the method comprising contacting a somatic cell with at least one of the recombinant circular RNAs described herein and maintaining the cell under conditions under which a reprogrammed iPSC is obtained.