Targeted Genetic Manipulation in Cells Using Single-Stranded HDR
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Solution Overview
Problem
Current treatments for CTLA4 haploinsufficiency, such as IV or subcutaneous injections of abatacept, are limited by the risk of worsening the disease due to disrupting the affected gene, and existing genome editing methods struggle with low efficiency and yield in inserting exogenous sequences to correct disease-causing mutations in autosomal dominant genes like CTLA4.
Innovation Solution
A method involving the insertion of an exogenous partial open reading frame (ORF) of an autosomal dominant gene into an intronic target region using a single-stranded DNA template and a targeted nuclease, guided by a specific guide RNA, and enhanced with small molecule inhibitors to improve efficiency and yield, resulting in a modified gene free of disease-causing mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If existing genome editing methods are used to insert exogenous sequences to correct disease-causing mutations, then gene correction is achieved, but the efficiency and yield are low
Solution Approach 1:
The patent changes the physical state of the DNA template from double-stranded to single-stranded, which fundamentally alters the parameters of the homology-directed repair process. This parameter change enables significantly higher correction efficiency (>80-90%) while maintaining reliability, as the single-stranded template is more efficiently incorporated into the genome during HDR without the complications of double-stranded DNA processing
Solution Approach 2:
The patent introduces small molecule inhibitors as intermediaries that modulate the cellular DNA repair machinery. These inhibitors temporarily suppress competing repair pathways (such as NHEJ), allowing the homology-directed repair process to dominate and achieve high efficiency. The inhibitors act as mediators between the exogenous single-stranded DNA template and the cellular repair apparatus
2Manufacturing precision
If targeted nuclease and guide RNA are used for precise gene editing, then mutation correction is achieved, but the insertion efficiency remains low
Solution Approach 1:
The patent changes the form of the donor DNA from double-stranded to single-stranded, which fundamentally improves the kinetics and efficiency of homology-directed repair. The single-stranded template can more readily invade the heteroduplex DNA formed at the double-strand break site, leading to dramatically improved insertion efficiency while maintaining the precision targeted by the guide RNA and nuclease
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 ultra-high HDR efficiencies (>80-90%) for gene correction, enabling effective treatment of CTLA4 haploinsufficiency and other immunodeficiencies, with potential for large-scale non-viral CAR-T cell manufacturing.
Implementation Method 1
a targeted nuclease that creates an insertion site in the intronic target region
Implementation Method 2
a guide RNA that specifically hybridizes to the intronic target region
Implementation Method 3
insertion of the exogenous partial ORF of the autosomal dominant gene into the intronic target region results in a modified autosomal dominant gene
Data Source
AI summary
Methods for editing the genome of cells such as T cells and hematopoietic stem cells are disclosed. The methods include inserting a nucleic acid sequence of an exogenous partial open reading frame (ORF) of an autosomal dominant gene (e.g., CTLA4) into an intronic target region of an endogenous autosomal dominant gene in the cell, wherein the endogenous autosomal dominant gene comprises one or more disease-causing mutations; the exogenous partial ORF of the autosomal dominant gene is free of disease-causing mutations; and insertion of the exogenous partial ORF of the autosomal dominant gene into the intronic target region results in a modified autosomal dominant gene that encodes a protein which is free of disease-causing mutations. Methods for treating haploinsufficiency and methods for increasing gene editing efficiency are also described.