Enabling correction of skeletal muscle genetic disorders
The aim of this project was to develop methodologies for robust, and safe in vivo gene editing in skeletal muscle tissue. The project has provided a valuable tool set for researchers around the world to perform gene targeting in skeletal muscle, enabling the development of personalised gene therapies for genetic muscle disease and metabolic diseases.
Most primary cell types have developed strong defensive mechanisms that prevent efficient manipulation of their genome. A proprietary technology has been developed called induced Transduction by Osmocytosis and Propanebetaine (iTOP), a unique method for the intracellular delivery of (large) bioactive molecules that is conceptually very different from other methods. iTOP is vastly superior in its ability to transduce primary cells, its unparalleled high efficiency of cell transduction, the narrow control over dosage and timing of the delivered protein and the non‐integrating nature of protein manipulation, improving safety and minimizing off‐target effects. iTOP technology was reported in a publication in the prestigious journal Cell last year (D’Astolfo et al., 2015 Cell, 161:674690).
The advantages of iTOP gene editing were quickly recognised by the field and recently highlighted in an opinion article in the leading journal Nature Methods (Nature Methods 12, 602 (2015) doi:10.1038/nmeth.3464). The iTOP transduction technology developed in the Hubrecht Institute provides an effective backdoor into these cells and allows efficient intracellular delivery of recombinant proteins into virtually any cell type. However, the iTOP reagent needs to be optimised and validated for (clinically relevant) use in vivo. The iTOP reagent was adapted to make it compatible with application in vivo.
The original published reagent contained several components of animal origin, which cannot be applied in a patient setting. Reformulation and re‐optimisation by the consortium partner NTrans Technologies has resulted in a fully synthetic reagent, which in addition, displays higher transduction efficiency and lower cell toxicity. At the same time, it enabled to demonstrate successful in vivo transduction of Cre protein as well as CRISPR/Cas9 in skeletal muscle fibers of mTmG reporter mice. Current work is aimed at the development of CRISPR strategies for the targeted functional repair of disease‐relevant genes, such as the Dystrophin gene, which plays a role in the pathogenesis of Duchenne Muscular Dystrophy.