CRISPR Gene Editing of Primary Human Muscle Stem Cells
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Solution Overview
Problem
Current methods for gene repair in muscle stem cells are limited, particularly for muscular dystrophy patients, as they often require immunosuppression, have low editing efficiency, and may lead to off-target effects or insertional mutagenesis, making them unsuitable for long-term therapeutic effects.
Innovation Solution
An ex vivo method using CRISPR/Cas-based tools for targeted gene editing of primary human muscle stem cells, delivered via plasmids, mRNA, or recombinant proteins, achieving high efficiency (>90%) without the need for selection markers, and maintaining regenerative capacity and immune compatibility.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If allogeneic transplantation of myoblasts or mesangioblasts is used, then cell replacement therapy can be provided, but immunosuppression is required and clinical benefit has not been achieved
Solution Approach 1:
The invention segments the therapy into two distinct approaches: allogeneic transplantation using universal donor cells and autologous transplantation using genetically corrected patient-specific cells. This segmentation allows selection of the most appropriate approach based on individual patient needs and availability of suitable donor cells.
Solution Approach 2:
The invention uses gene editing tools (CRISPR/Cas9, base editors, prime editors) as intermediaries to correct genetic defects in autologous muscle stem cells ex vivo. These editing tools serve as mediators that enable genetic correction without requiring immunosuppression, bridging the gap between genetic defect and functional restoration.
2Quantity of substance
If iPSC-derived muscle cells are used for transplantation, then unlimited cell source is available, but quality and safety criteria are not met
Solution Approach 1:
The invention performs preliminary genetic correction of muscle stem cells ex vivo before transplantation. By correcting the genetic defect in the stem cells prior to implantation, the therapy ensures long-term production of healthy muscle tissue without the safety concerns associated with iPSC differentiation and tumorigenicity.
Solution Approach 2:
The corrected muscle stem cells serve themselves by continuously producing healthy muscle tissue in vivo. The genetically corrected stem cells maintain their regenerative capacity and self-renewal properties, providing sustained therapeutic effect without requiring repeated administrations or complex differentiation protocols.
3Manufacturing precision
If traditional gene editing methods are used, then gene repair can be attempted, but off-target effects and insertional mutagenesis occur
Solution Approach 1:
The invention replaces traditional mechanical DNA cutting and integration methods with precision-based editing mechanisms. CRISPR/Cas9 provides targeted DNA cleavage at specific sequences, while base editors and prime editors enable direct chemical modification of DNA bases without double-strand breaks, eliminating the need for donor DNA templates and reducing off-target effects.
Solution Approach 2:
The invention changes the fundamental parameters of gene editing by transitioning from non-specific to highly specific targeting mechanisms. The use of guide RNAs with precise complementarity to target sequences, combined with optimized Cas9 variants and editing windows, achieves unprecedented specificity and reduces off-target effects to minimal levels.
4Adaptability or versatility
If MuSC are isolated and expanded from human muscle biopsy, then autologous transplantation is possible, but genetic defect correction is required before reimplantation
Solution Approach 1:
The invention extracts the genetic defect from the muscle stem cells through targeted editing. By removing or correcting the specific mutated sequence using CRISPR/Cas9 or base editing, the pathological element is eliminated while preserving the cell's regenerative function, enabling safe autologous transplantation.
Solution Approach 2:
The invention performs preliminary genetic correction of muscle stem cells ex vivo before transplantation. By correcting the genetic defect in the stem cells prior to implantation, the therapy ensures long-term production of healthy muscle tissue without the safety concerns associated with introducing defective genes.
Data Source
AI summary
It is provided a method for gene repair in primary human muscle stem cells (satellite cells) in vitro comprising the following steps: providing a sample of an isolated muscle-fiber containing tissue sample collected from at least one patient with a monogenic muscle disease, wherein the monogenic muscle disease is caused by at least one mutation in at least one gene encoding for at least one muscle protein; isolating and cultivating primary stem cells from said muscle-fiber containing tissue sample, and correcting the at least one mutation in the at least one gene encoding for at least one muscle protein in the cultivated primary stem cells by targeted modification of the at least one mutation by gene editing using CRISPR/Cas-based tools.