Genomic DNA Modification via Donor DNA and Digesting Agent
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Solution Overview
Problem
Current genome engineering approaches for gene therapy are inefficient, toxic, and lack specificity, introducing harmful DNA sequences and triggering immunogenicity, limiting their effectiveness in site-directed genomic DNA modification.
Innovation Solution
A method involving contacting cells with an activating composition and transfecting them with a composition comprising donor DNA and a DNA digesting agent, where the donor DNA has a homologous region and a sequence modification region, allowing for site-specific sequence modification of genomic DNA, including the integration of transgenes, using techniques like CRISPR and electroporation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current genome engineering approaches are used for gene therapy, then gene transfer can be achieved, but the efficiency of repair or editing is very low and harmful DNA sequences may be introduced
Solution Approach 1:
The donor DNA is segmented into two distinct regions: a homologous region for precise targeting and a sequence modification region for the desired genetic change. This segmentation allows the DNA digesting agent to create a break at the target site while the homologous region guides precise repair, separating the functions of targeting and modification to improve efficiency and reduce harmful effects
Solution Approach 2:
The homologous region acts as an intermediary between the DNA digesting agent's break site and the sequence modification region. It facilitates precise alignment and repair by providing complementary sequences that guide the repair mechanism to the exact target location, ensuring high efficiency and specificity while preventing random integration of harmful sequences
2Ease of operation
If viral vectors are used for gene transfer, then gene therapy can be delivered, but viral sequences are introduced into the host which may trigger immunogenicity
Solution Approach 1:
The harmful viral sequences are completely extracted from the gene delivery system. Instead of using viral vectors, the invention employs non-viral donor DNA combined with a DNA digesting agent, eliminating the source of immunogenicity while maintaining the ability to deliver gene therapy through electroporation or other non-viral transfection methods
Solution Approach 2:
The invention uses disposable, non-viral donor DNA molecules that are introduced into cells temporarily to perform the desired genetic modification. These DNA molecules do not integrate into the host genome permanently and are eventually degraded, avoiding the long-term immunogenicity problems associated with viral vectors while still achieving effective gene transfer
3Object-generated harmful factors
If non-viral methods are used for gene therapy, then immunogenicity is reduced, but efficiency is low or toxicity is high
Solution Approach 1:
The DNA digesting agent is introduced first to create a specific break at the target genomic location before the donor DNA is introduced. This preliminary action prepares the cell's repair machinery to be highly active and receptive to the incoming donor DNA, dramatically improving the efficiency of non-viral gene therapy while maintaining low immunogenicity
Solution Approach 2:
The invention changes the temporal parameters of DNA introduction by using sequential delivery: first the DNA digesting agent to create breaks, then the donor DNA to be repaired. This time-separated approach allows each component to perform its function optimally, achieving high efficiency without the toxicity or low efficiency problems of traditional simultaneous non-viral methods
4Manufacturing precision
If current genome engineering methods are used, then some genomic modification can be achieved, but specificity is lacking and random integration occurs
Solution Approach 1:
The donor DNA exhibits local quality differentiation with a homologous region designed to match the specific target sequence and a sequence modification region containing the desired change. This local specificity ensures that the DNA digesting agent's break is repaired only at the intended target site, preventing random integration while achieving precise genomic modification
Solution Approach 2:
The homologous region provides a feedback mechanism by base-pairing with the complementary sequence at the break site created by the DNA digesting agent. This molecular feedback ensures that repair occurs only at the correct location where homology exists, dramatically improving specificity and eliminating random integration events
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 method enhances the efficiency and specificity of genomic DNA modification, reducing toxicity and random integration, enabling stable and targeted gene editing for therapeutic applications.
Implementation Method 1
a DNA digesting agent
Implementation Method 2
One region is a homologous region comprising nucleic acid sequence homologous to the target genomic DNA region
Data Source
AI summary
Compositions and methods concern the sequence modification of an endogenous genomic DNA region. Certain aspects relate to a method for site-specific sequence modification of a target genomic DNA region in cells comprising: contacting the cells with an activating composition; transfecting the cells with a transfection composition comprising (a) donor DNA and (b) a DNA digesting agent; wherein the donor DNA comprises: (i) a homologous region comprising nucleic acid sequence homologous to the target genomic DNA region; and (ii) a sequence modification region; and wherein the genomic DNA sequence is modified specifically at the target genomic DNA region.