SCN10A Gene Editing With CRISPR-Cas9 for Reproducible Pain Treatment
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Solution Overview
Problem
Current genome engineering technologies for altering the SCN10A gene are limited by random insertion and lack of reproducibility, posing challenges in developing safe and effective treatments for related disorders.
Innovation Solution
A single-molecule guide RNA (sgRNA) and S. pyogenes Cas9 endonuclease are introduced into cells to create precise single-strand or double-strand breaks in the SCN10A gene, enabling permanent insertions, deletions, or mutations to reduce or eliminate SCN10A gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If random insertion technologies are used to alter the SCN10A gene, then gene modification can be achieved, but the precision and reproducibility of the modification are poor
Solution Approach 1:
The guide RNA is divided into distinct functional domains (spacer sequence, repeat sequence, and optional extension sequences) that can be independently optimized. This segmentation allows precise targeting of the SCN10A gene while maintaining reproducibility across different cells and treatments.
Solution Approach 2:
The CRISPR-Cas9 system acts as an intermediary between the guide RNA and the SCN10A gene. The Cas9 endonuclease mediates the precise cutting of the target gene based on guide RNA direction, enabling accurate and reproducible gene modification without random insertion.
2Productivity
If multiple treatments are required to achieve gene editing, then the treatment complexity increases, but a single treatment is desired for permanent cure
Solution Approach 1:
The guide RNA is designed with extended sequences that facilitate stable complex formation with Cas9 before target engagement. This preliminary optimization ensures efficient gene editing in a single treatment, eliminating the need for multiple sequential therapies.
Solution Approach 2:
The CRISPR-Cas9 system with optimized guide RNA serves multiple functions simultaneously: it guides Cas9 to the target site, facilitates DNA cutting, and enables permanent gene modification. This multi-functionality allows a single treatment to achieve complete and lasting therapeutic effect.
3Duration of action of stationary object
If genome engineering is used to permanently alter the SCN10A gene, then the therapeutic effect can be lasting, but safety concerns arise from potential off-target effects
Solution Approach 1:
The guide RNA is designed with a spacer sequence that is highly specific to the SCN10A gene target site. This localized precision ensures that Cas9 cuts only at the intended location, minimizing off-target effects while maintaining permanent therapeutic benefit.
Solution Approach 2:
The extended sequences in the guide RNA structure provide feedback mechanisms that enhance the stability and specificity of the Cas9-guide RNA complex. This feedback ensures accurate target recognition and reduces the likelihood of off-target cutting, enabling safe permanent gene modification.
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 allows for a single treatment to permanently alter the SCN10A gene, potentially curing related disorders by reducing or eliminating gene function, thereby addressing the limitations of existing technologies.
Implementation Method 1
one or more S. pyogenes Cas9 endonuclease or one or more polynucleotide encoding the one or more S. pyogenes Cas9 endonuclease; to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the SCN10A gene
Data Source
Figure 1A~1B
Figure 2A
Figure 2B
AI summary
The present application provides materials and methods for treating a patient with one or more conditions associated with SCN10A whether ex vivo or in vivo. In addition, the present application provides materials and methods for editing and/or modulating the expression of SCN10A gene in a cell by genome editing.