Coding Sequence Replacement for Precise Endogenous Gene Editing
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Solution Overview
Problem
Current gene therapy approaches for severe combined immunodeficiency (SCID) using viral vectors face safety concerns such as transgene integration into tumor-suppressor loci, incomplete phenotypic correction, toxicity, and dysregulated hematopoiesis, particularly for tightly regulated genes like RAG1/2, necessitating a safer and more precise method for gene editing.
Innovation Solution
A system for gene editing that replaces a portion of the coding sequence of endogenous genes, such as RAG1/2, using a CRISPR/Cas9 system to introduce targeted double-strand breaks and a replacement nucleic acid molecule with homology arms to facilitate precise homology-directed repair (HDR), ensuring accurate integration and expression of a transgene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If viral vectors are used for transgene delivery in gene therapy, then gene correction can be achieved, but safety concerns arise including transgene integration into tumor-suppressor loci, incomplete phenotypic correction, toxicity, and dysregulated hematopoiesis
Solution Approach 1:
The patent segments the coding sequence into multiple exons and targets specific exons for replacement rather than delivering the entire gene. This allows precise modification of disease-causing regions while preserving functional elements, reducing the risk of disrupting tumor-suppressor loci during integration.
Solution Approach 2:
The invention applies local quality by replacing only specific coding sequence regions (exons) containing disease-causing variants while maintaining the rest of the endogenous gene structure and regulatory elements. This localized approach ensures that only the problematic regions are modified, avoiding widespread integration issues.
2Reliability
If complete gene replacement is performed, then functional correction is achieved, but disruption of genomic regulatory elements and chromatin structure occurs
Solution Approach 1:
The patent segments the gene into coding sequences (exons) and non-coding regulatory regions, and only replaces the coding segments. This preserves the genomic regulatory elements and chromatin structure that control gene expression, while still achieving functional correction of the coding regions.
Solution Approach 2:
The invention extracts and replaces only the problematic coding sequence portions (exons) containing disease-causing variants, while leaving the regulatory elements and chromatin structure intact. This selective extraction approach maintains genomic stability while achieving functional correction.
3Productivity
If viral vectors are used for ex vivo editing of CD34+ HSPCs, then immune system reconstitution is achieved, but insertional mutagenesis and leukemic transformation occur
Solution Approach 1:
The patent segments the gene delivery approach by using targeted exon replacement rather than complete gene replacement via viral vectors. This reduces the amount of foreign DNA that must be integrated, thereby reducing the risk of insertional mutagenesis while still achieving functional correction for immune system reconstitution.
Solution Approach 2:
The invention replaces the mechanical integration mechanism of viral vectors with a targeted exon replacement approach that utilizes the cell's own repair mechanisms. This substitution eliminates the random integration process that causes insertional mutagenesis while maintaining productive immune system reconstitution.
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 achieves precise gene correction with minimal disruption to genomic regulatory elements, reducing the risk of genomic instability and lymphocyte malignancy, and enables functional immune cell development with appropriate gene expression levels.
Implementation Method 1
at least one genome editing reagent designed for generating a double strand break (DSB) in a region spanning the gene portion sequence
Implementation Method 2
a replacement nucleic acid molecule comprising a coding sequence replacement (CDSR) construct designed for serving as a template for homology-directed repair (HDR) triggered by the DSB
Data Source
AI summary
The present invention provides a system and a method for editing of an endogenous gene by replacing a gene portion sequence thereof comprised in a single exon with a transgene sequence, the system comprising at least one genome editing reagent designed for generating a double strand break (DSB) in a region spanning the gene portion sequence; and a replacement nucleic acid molecule comprising a coding sequence replacement (CDSR) construct designed for serving as a template for homology-directed repair (HDR) triggered by the DSB.