3E10-Potentiated Gene Editing for Higher Specificity
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing gene editing technologies, such as CRISPR/Cas9, suffer from low efficiency and high off-target effects, particularly in primary stem cells, limiting their effectiveness in correcting genetic disorders.
Innovation Solution
Combining triplex-forming oligonucleotides or CRISPR systems with a cell-penetrating anti-DNA antibody, like 3E10, to enhance genomic modification frequency and reduce off-target effects by modulating endogenous DNA repair pathways, specifically inhibiting RAD51 activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If CRISPR/Cas9 or targeted nucleases are used for gene editing, then ease of use and reagent design are improved, but off-target cleavage increases
Solution Approach 1:
The patent introduces triplex-forming oligonucleotides (TFOs) as intermediary molecules that bind to the target DNA sequence and recruit endogenous DNA repair machinery, serving as a mediator between the desired editing outcome and the cellular repair systems, thereby avoiding direct nuclease cleavage and reducing off-target effects
Solution Approach 2:
The patent leverages the cell's own endogenous DNA repair systems (homologous recombination and non-homologous end joining) to perform the gene editing function, allowing the cellular machinery to serve itself rather than requiring externally introduced active nucleases, thus eliminating off-target cleavage while maintaining editing capability
2Object-generated harmful factors
If triplex-forming oligonucleotides are used for gene editing, then off-target effects are reduced, but editing efficiency decreases
Solution Approach 1:
The patent modifies the chemical structure of oligonucleotides by incorporating peptide nucleic acid (PNA) backbones instead of traditional phosphodiester backbones, changing the physical and chemical parameters such as binding affinity, stability, and cellular uptake characteristics, thereby improving editing efficiency while maintaining specificity
Solution Approach 2:
The patent creates composite molecular structures by combining PNA backbones with DNA-like base pairs to form PNA-DNA hybrid triplexes, utilizing the advantageous properties of both PNA (stability, binding affinity) and DNA (complementarity, specificity) to achieve high efficiency and low off-target effects simultaneously
3Productivity
If donor DNA is co-delivered with triplex-forming oligonucleotides, then gene modification frequency increases, but delivery complexity increases
Solution Approach 1:
The patent combines multiple therapeutic agents (triplex-forming oligonucleotides and donor DNA) into a single delivery vehicle or formulation, merging the editing function and the template function into one coordinated delivery system, thereby increasing gene modification frequency while managing delivery complexity through unified administration
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
Enhances the frequency of on-target gene modification while minimizing off-target effects, improving the efficacy of gene editing in both cultured cells and in vivo models.
Implementation Method 1
modulating endogenous DNA repair pathways, specifically inhibiting RAD51 activity
Implementation Method 2
triplex-forming oligonucleotides
Data Source
AI summary
Compositions for improved gene editing and methods of use thereof are disclosed. In a preferred method, gene editing involves use of a cell-penetrating anti-DNA antibody, such as 3E10, as a potentiating agent to enhance gene editing by nucleases and triplex forming oligonucleotides. Genomic modification occurs at a higher frequency when cells are contacted with the potentiating agent and nuclease or triplex forming oligonucleotide, as compared to the absence of the potentiating agent. The methods are suitable for both ex vivo and in vivo approaches to gene editing and are useful for treating a subject with a genetic disease or disorder. Nanoparticle compositions for intracellular delivery of the gene editing compositions are provided and are particularly advantageous for use with in vivo applications.