Primary Cell Genome Editing With Non-Viral HDR Templates
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Solution Overview
Problem
Existing methods for genome editing in primary cells, such as primary T cells, are inefficient and often require the use of viral vectors, which can be risky and impractical for certain applications.
Innovation Solution
A method using non-viral DNA templates and a nuclease to introduce a knock-in cassette into the genome of primary cells through homology-directed repair (HDR), ensuring high efficiency and integration of an exogenous coding sequence for a gene product of interest while maintaining expression of an essential gene required for cell survival.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If viral vectors are used for genome editing in primary cells, then editing efficiency may be improved, but safety risks and practicality deteriorate
Solution Approach 1:
The patent extracts and removes the viral vector component from the genome editing system, replacing it with non-viral DNA templates. This eliminates the safety risks associated with viral vectors while maintaining the ability to achieve efficient gene knock-in in primary cells through homology-directed repair mechanisms
Solution Approach 2:
The patent employs transient, non-integrating DNA templates that serve their purpose during the editing process and are then discarded, rather than using persistent viral vectors. This approach reduces long-term safety concerns while achieving the necessary editing efficiency through controlled homology-directed repair
2Reliability
If non-viral DNA templates are used for genome editing, then safety risks are reduced, but editing efficiency deteriorates
Solution Approach 1:
The patent optimizes parameters such as DNA template design, homology arm length, and nuclease timing to maximize editing efficiency while maintaining safety. By carefully controlling these parameters, the system achieves high knock-in efficiency without requiring viral vectors
Solution Approach 2:
The patent introduces homology-directed repair as an intermediary mechanism that facilitates efficient gene knock-in using non-viral DNA templates. This intermediary process enables the cell to accurately integrate exogenous DNA sequences without the need for viral vectors, thereby maintaining both safety and efficiency
3Adaptability or versatility
If essential genes are targeted for editing, then gene function can be modified, but cell survival may be compromised
Solution Approach 1:
Instead of disrupting essential genes to achieve editing, the patent inverts the approach by using homology-directed repair to precisely insert genetic material into essential genes, thereby modifying their function while preserving their essential nature. This inversion allows functional modification without compromising cell survival
Solution Approach 2:
The patent applies local quality changes by introducing exogenous coding sequences at specific locations within essential genes through homology-directed repair. This allows precise modification of gene function at targeted locations while maintaining the overall integrity and survival function of the essential gene
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 approach achieves high editing efficiency, with over 60% of viable cells being genome-edited and expressing both the essential gene product and the gene product of interest, while minimizing the risk associated with viral vectors.
Implementation Method 1
a nuclease that causes a break within an endogenous coding sequence of an essential gene
Implementation Method 2
the knock-in cassette is integrated into the genome of the cell by homology-directed repair (HDR) of the break
Data Source
AI summary
Strategies, systems, compositions, and methods for genetically modifying cells to include one or more loss-of-function modifications and/or to include one or more gain-of-function modifications, as well as modified cells (and compositions of such cells) that include one or more loss-of-function modifications and/or that include one or more gain-of-function modifications, are described. In certain aspects, such modified cells include at least one gain-of-function modification within a coding region of an essential gene.