DNA-PKcs and 53BP1 Inhibitor Composition for HDR Gene Editing
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Solution Overview
Problem
Existing cell therapies face challenges such as high cell death rates and low transduction efficiency due to genetic manipulations, particularly in gene editing methods like CRISPR-Cas, which cause excessive DNA double-strand breaks leading to non-homologous end joining (NHEJ) events, reducing the efficiency of homology-directed repair (HDR).
Innovation Solution
A composition comprising a DNA-dependent protein kinase catalytic subunit inhibitor (e.g., AZD-7648, M3814) and a 53BP1 inhibitor (e.g., i53BP1) is used to inhibit NHEJ pathways and enhance HDR events, increasing genome editing efficiency while maintaining cell viability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If gene editing methods like CRISPR-Cas are used to cause DNA double-strand breaks, then genome editing efficiency can be improved, but excessive cell death occurs due to toxicity from non-homologous end joining (NHEJ) events
Solution Approach 1:
The patent applies preliminary anti-action by pre-treating cells with DNA-PKcs and 53BP1 inhibitors before gene editing to block the NHEJ pathway in advance. This prevents the harmful NHEJ repair mechanism from acting on DNA double-strand breaks, thereby reducing cell death while allowing HDR to proceed effectively.
Solution Approach 2:
The patent converts the harmful effect of DNA-PKcs and 53BP1 in NHEJ pathway into a beneficial outcome by selectively inhibiting these proteins. This suppression of NHEJ toxicity transforms what would normally be a cell-killing mechanism into a condition that favors HDR-mediated precise gene editing with improved cell survival.
2Productivity
If the amount of nucleic acid used in genetic manipulations is increased to improve transduction rates, then genetic manipulation efficiency can be improved, but cell death increases due to toxicity
Solution Approach 1:
The patent applies parameter changes by modifying the cellular environment through inhibitor treatment, which changes the toxicity threshold parameter. This allows higher nucleic acid amounts to be used for transfection without reaching the toxic threshold, thereby improving transduction efficiency while maintaining cell survival.
3Speed
If non-homologous end joining (NHEJ) pathways are activated to repair DNA double-strand breaks, then DNA repair speed is improved, but homology-directed repair (HDR) efficiency decreases
Solution Approach 1:
The patent applies the taking out principle by selectively removing or inhibiting the DNA-PKcs and 53BP1 components from the NHEJ pathway. This extraction of key NHEJ proteins prevents NHEJ from competing with HDR, allowing HDR to become the dominant repair mechanism while maintaining efficient DNA repair through the inhibited NHEJ pathway's partial functionality.
Solution Approach 2:
The patent applies inversion by reversing the natural repair pathway preference. Instead of allowing cells to naturally favor NHEJ for speed, the inhibitors reverse this preference by blocking NHEJ, thereby inverting the repair pathway hierarchy to favor HDR, which normally would be slower but provides precise editing.
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
The combination of DNA-PKcs and 53BP1 inhibitors significantly enhances HDR-mediated knock-in events by 17.5-fold, reducing toxicity and improving cell survival, making gene editing more efficient and viable.
Implementation Method 1
a DNA-dependent protein kinase catalytic subunit inhibitor
Implementation Method 2
a 53BP1 inhibitor
Data Source
Figure 1
Figure 2a~2b
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AI summary
The present invention relates to a composition comprising a DNA-dependent protein kinase catalytic subunit inhibitor and a 53BP1 inhibitor. The present invention also relates to a composition comprising a DNA-dependent protein kinase catalytic subunit inhibitor and a 53BP1 inhibitor for use in medicine. Furthermore, the present invention relates to a gene editing enhancing composition comprising a DNA-dependent protein kinase catalytic subunit inhibitor and a 53BP1 inhibitor. Besides, the present invention relates to a gene editing method comprising the sequential steps of: (i) enriching isolated cells, followed by activating said isolated cells by pre-determined surface antigens; (ii) adding a composition according to the invention to said cells simultaneously to transfecting said cells two days after step (i); (iii) adding a stimulant to said cells five days after step (i); (iv) analyzing the genome editing efficiency and viability of said cells by fluorescent-activated cell sorting. Moreover, the present invention relates to an engineered cell obtained by the gene editing method of the present invention. Additionally, the present invention relates to the use of the composition comprising a DNA-dependent protein kinase catalytic subunit inhibitor and a 53BP1 inhibitor in gene editing.