Microduplication Gene Repair Using MMEJ in Post-Mitotic Cells
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Solution Overview
Problem
Current genome editing methods, particularly those using programmable nucleases, are inefficient in correcting microduplication mutations in post-mitotic differentiated cell types such as neurons and muscle cells, which are common in genetic disorders, due to the inefficiency of the Homology Directed Repair (HDR) pathway.
Innovation Solution
The use of a programmable nuclease to create a double-strand break within a microduplication, inducing the microhomology mediated end joining (MMEJ) DNA repair pathway, which corrects the microduplication mutation to a wild-type sequence without the need for exogenous donor DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If Homology Directed Repair (HDR) pathway is used to correct microduplication mutations, then precise sequence correction can be achieved, but the repair efficiency is extremely low in post-mitotic differentiated cell types
Solution Approach 1:
The invention changes the DNA repair pathway parameter from HDR to MMEJ by designing the donor DNA with microhomology sequences (2-25 bp) that match the sequences flanking the microduplication. This parameter change enables efficient repair in post-mitotic cells while maintaining precise correction of the mutation
Solution Approach 2:
The invention uses a simplified copying mechanism where microhomology sequences from the flanking regions are copied to join the broken DNA ends, eliminating the need for complex HDR machinery. The donor DNA serves as a template only for the microhomology regions, not for the entire correction sequence
2Manufacturing precision
If exogenous donor DNA is provided for HDR-based correction, then precise sequence re-writing can be achieved, but the delivery complexity and immunogenicity increase significantly
Solution Approach 1:
The invention extracts only the essential microhomology sequences from the donor DNA and places them at the ends, removing the need for extensive homology regions. This simplifies the donor DNA structure and reduces delivery complexity while maintaining correction precision
Solution Approach 2:
Instead of providing a complete donor DNA template for HDR, the invention inverts the approach by providing a donor that primarily consists of flanking microhomology sequences that enable the cell's own MMEJ pathway to perform the correction, reducing immunogenicity and delivery burden
3Productivity
If microhomology sequences of 1-40 nucleotides are used in MMEJ repair, then efficient correction of microduplications is achieved, but the risk of off-target effects increases
Solution Approach 1:
The invention applies local quality by placing microhomology sequences specifically at the 5' and 3' ends of the donor DNA that flank the correction site. These localized microhomology regions guide the MMEJ repair precisely at the target location, improving efficiency while the specificity of the nuclease guide RNA minimizes off-target effects
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 efficiently and precisely corrects microduplication mutations in patient-derived cell lines, including TCAP and HPS1, restoring wild-type sequences and reducing disease symptoms, with potential applicability to over 100 genetic disorders associated with microduplications.
Implementation Method 1
the creation of a double stranded break within a microduplication by a programmable nuclease protein
Implementation Method 2
induces the microhomology mediated end joining (MMEJ) DNA repair pathway
Data Source
AI summary
The present invention is directed to the filed of gene therapy. In particular, compositions and methods are disclosed that repair gene microduplication mutations by reversion to a wild type sequence. For example, the creation of a double stranded break by a programmable nuclease protein within a microduplication induces the microhomology mediated end joining DNA repair pathway that in the process of DNA repair removes the microduplication mutation and restores the wild type sequence.