DNA Minicircles for Genome Engineering Delivery
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Solution Overview
Problem
Current methods for delivering nucleic acids for genome engineering, such as plasmid-based systems, are often toxic to cells and inefficient due to the presence of bacterial DNA sequences that trigger immune responses and are not optimally designed for targeted gene modification.
Innovation Solution
The use of DNA minicircles (MCs) for delivering nucleic acids, which are devoid of bacterial sequences and can carry nuclease systems like CRISPR/Cas, zinc finger nucleases, or TALENs, to facilitate targeted cleavage and integration of exogenous sequences into specific genomic locations, reducing toxicity and improving efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If plasmid-based delivery systems are used to deliver nucleic acids for genome engineering, then the delivery can be achieved, but the cell toxicity increases and delivery efficiency decreases due to bacterial DNA sequences triggering immune responses
Solution Approach 1:
The patent removes bacterial DNA sequences (origin of replication, antibiotic resistance genes, CpG motifs) from the plasmid backbone to create minicircle DNA. This extraction eliminates the harmful immune-triggering elements while retaining the essential gene delivery function, thereby reducing cell toxicity and improving delivery efficiency to target cells.
2Productivity
If standard plasmids are used for gene therapy, then gene delivery can be achieved, but the inflammatory response increases due to unmethylated CpG motifs from bacterial DNA
Solution Approach 1:
The patent converts the harmful bacterial DNA sequences into a benefit by completely removing them to create minicircle DNA. The absence of CpG motifs and other bacterial elements eliminates the inflammatory response triggered by the immune system, while the compact minicircle structure enhances nuclear entry and gene delivery efficiency to therapeutic levels.
3Quantity of substance
If plasmid DNA is transfected into human cells for genome engineering, then the nucleic acid delivery can be achieved, but the process becomes inefficient and toxic due to foreign DNA recognition
Solution Approach 1:
The patent extracts and removes the bacterial plasmid backbone sequences that are recognized as foreign by mammalian cells. The resulting minicircle DNA lacks these immunogenic elements, allowing efficient transfection into human cells without triggering foreign DNA recognition pathways, thereby improving both delivery quantity and transfection ease.
Data Source
AI summary
Disclosed herein are methods and compositions for delivery of engineered nucleases and donor molecules into the genome of a cell.