Synthetic RNA Transfection for Safer Cell Reprogramming
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Solution Overview
Problem
Current methods for transfecting cells, reprogramming cells, and gene editing are inefficient, carry risks of mutation, and require the use of exogenous DNA or animal-derived materials.
Innovation Solution
The use of synthetic RNA molecules containing non-canonical nucleotides, such as pseudouridine and 5-methylcytidine, which are produced by in vitro transcription and can encode gene-editing proteins, along with a medium containing treated albumin and hydrocortisone, to facilitate high-efficiency transfection, reprogramming, and gene editing without the need for exogenous DNA or animal-derived components.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If exogenous DNA or animal-derived materials are used for transfection and gene editing, then transfection efficiency can be achieved, but the risk of mutation increases and cell viability decreases
Solution Approach 1:
The patent extracts and eliminates the harmful components (exogenous DNA and animal-derived materials) from the transfection process while retaining the essential function of delivering genetic material. This is achieved by using only chemically synthesized RNA components, removing the source of mutations and toxicities associated with traditional transfection reagents.
Solution Approach 2:
The patent changes the chemical parameters of the transfection reagents by using chemically synthesized RNA with modified nucleotides instead of exogenous DNA or animal-derived materials. This parameter change reduces immunogenicity and toxicity while maintaining transfection efficiency, directly addressing the harmful effects of traditional methods.
2Productivity
If traditional transfection reagents are used, then nucleic acid delivery can be achieved, but cell viability and safety are compromised
Solution Approach 1:
The patent uses chemically synthesized RNA molecules that are transient and do not integrate into the genome, replacing permanent transfection reagents with temporary, easily degradable carriers. This approach maintains delivery efficiency while improving safety, as the synthetic RNA does not persist in the cell and can be cleared without long-term effects.
Solution Approach 2:
The patent employs composite RNA structures combining different nucleotide types (including modified nucleotides like pseudouridine and 5-methylcytidine) to create a material that is both efficiently taken up by cells and biologically safe. This composite approach optimizes both productivity and reliability by balancing delivery efficiency with reduced immunogenicity.
3Productivity
If serum-containing media are used for cell culture, then cell growth is promoted, but batch-to-batch variability and contamination risk increase
Solution Approach 1:
The patent extracts and removes serum components from the cell culture media, replacing them with chemically defined alternatives. This eliminates the source of batch-to-batch variability and contamination risk while maintaining the essential growth-promoting functions through precisely controlled supplementation with specific factors.
Solution Approach 2:
The patent changes the compositional parameters of the cell culture media by transitioning from complex, undefined serum-based formulations to precisely defined synthetic media compositions. This parameter change ensures consistent, reproducible results while eliminating contaminants, directly addressing the stability issue.
Data Source
AI summary
The present invention relates in part to nucleic acids encoding proteins, nucleic acids containing non-canonical nucleotides, therapeutics comprising nucleic acids, methods, kits, and devices for inducing cells to express proteins, methods, kits, and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics produced using these methods, kits, and devices. Methods for inducing cells to express proteins and for reprogramming and gene-editing cells using RNA are disclosed. Methods for producing cells from patient samples, cells produced using these methods, and therapeutics comprising cells produced using these methods are also disclosed.