Overcoming the problem of systemic delivery for ex vivo gene therapy of muscular dystrophy

Genetic diseases that affect widespread tissues like muscles or bones pose challenges for cell transplantation due to the need to target a large fraction of our body. Satellite cells, the canonical myogenic stem cells (Mauro et al. JCB 1961), do not migrate more than 1 or 2 mm away from the site of injection (Beauchamp et al. JCB 1999) so that numberless injections would be needed to target the majority of skeletal muscles. Mesoangioblasts (Mabs) are adventitial pericytes that can differentiate into skeletal muscle and cross the vessel wall in the presence of inflammation (Minasi et al. Development 2002), thus offering a potential route for systemic administration. In 2011, after encouraging results in animal models (Sampaolesi et al. Science 2003; Sampaolesi et al. Nature 2006), we conducted a clinical trial based upon repeated intra-arterial injections of donor Mabs from HLA-matched siblings. The trial proved safe (though with a vascular adverse event) but not efficacious (Cossu et al. EMBO Mol Med 2015). Thus, we developed a novel approach of ex vivo gene therapy, using autologous Mabs engineered to express a small nuclear RNA able to induce exon-skipping of exon 51 in the dystrophin gene. Since muscle fibres contain many nuclei, the small RNA diffuses into the resident neighbouring dystrophic nuclei within the fused fibres, thus amplifying dystrophin production to therapeutic levels (Galli et al. EMBO Mol Med 2024). Here we propose to overcome challenges in systemic delivery, thanks to the development and validation of a biocompatible, clinically-approved scaffold of laminin-coated polycaprolactone nanofibres that support efficient proliferation of mesoderm human cells. When transplanted in a subcutaneous pocket (a minimally invasive and repeatable procedure) of a DMD, immune deficient mouse, cells not only colonize the underlying dorsal muscle but also enter the general circulation through blood vessels rapidly grown into the scaffold; from there, they colonize all the muscles that we have analyzed including the diaphragm and the heart. We will optimize the experimental conditions such as time, number of cells, and eventual optimal number of subsequent implantations (aim 1), using both wt and DMD genetically corrected cells, at different cell numbers (aim 2); we will also test colonization of the heart (aim 3), measuring functional recovery of motility and cardiac function. If successful, this project will revolutionize cell delivery through the circulation, by eliminating the need for repeated, invasive arterial catheterizations that require general anesthesia and also fail to reach important muscles such as the diaphragm. Thus the success of this project would transform cell therapy for muscular dystrophies. Of notice this approach may subsequently be applied to other monogenic diseases of the mesoderm with a major impact on regenerative medicine.