Towards a new cell therapy for muscular dystrophy

Muscular dystrophies are heterogeneous diseases, characterized by primary wasting of skeletal muscle. Duchenne Muscular Dystrophy (DMD) is the most common muscular dystrophy and the second most common monogenic disorder, affecting 1/5000 newborn males. DMD is a severe disease, leading to wheelchair dependency, cardiac and respiratory failure, and premature death (Emery, Lancet 2002). In DMD, lack of dystrophin, a protein linking the cytoskeleton to the basal lamina, causes increased fragility of the sarcolemma. This results in focal or diffuse damage to the fiber during contraction (Blake et al. Physiol Rev 2002).

Damaged or dead fibers are repaired or replaced by satellite cells (Mauro, J Cell Biol 1961), the canonical myogenic stem cells of adult muscle. However, dystrophic satellite cells produce new fibers also prone to degeneration so that, after cycles of degeneration/regeneration, this cell population is exhausted, and the muscle tissue is replaced by connective and adipose tissue. At this stage any therapeutic intervention is likely to fail. Currently, corticosteroids represent the only consolidated therapy, but they only delay the progress of the disease and have significant side effects (Matthews et al. Cochrane Database Syst Rev 2016). However, drugs for the heart, assisted ventilation and improvement in patients care have ameliorated length and to a certain extent quality of life (Shah & Yokota Ther Adv Neurol Disord. 2023).

Despite almost forty years have gone by since the cloning of the dystrophin gene (Hoffman et al. Cell 1987), there is still no efficacious therapy. The cost for the NHS is progressively increasing with the increase of patient life and the need for more complex and expensive therapies, with a total burden of many hundred thousand GBP/patient. This cost could be eliminated by a successful therapy, in addition to the logistic and economic burden for the family and the possibility to return the patient to a normal and productive life.

Currently two major strategies are pursued by companies actively engaged in this field: 1. in vivo gene therapy mostly using Adeno-Associated Viral vectors (AAV) expressing either micro-dystrophin or gene editing tools to correct the mutation in vivo; 2. Antisense Oligonucleotides (AON) designed to skip the dystrophin exon carrying the mutation (Patterson et al. Eur J Pharmacol. 2023). Despite massive investments and many trials, none has reached significant clinical efficacy, and the first approach caused many severe adverse events, culminated with the death of three patients in three different trials (Duan et al. Mol Ther. 2023).

In this scenario, cell therapy, for long time considered “not efficacious”, is now gaining new momentum with the use of iPS cells and with adopting new strategies such as the one we developed, thanks to previous MRC support.

In the past, we had investigated systemic intra-arterial delivery of vessel-associated myogenic progenitors, named mesoangioblasts, and tested these therapeutic strategies in murine and canine models of muscular dystrophies (Sampaolesi et al. Science 2003; Sampaolesi et al. Nature 2006). Based on encouraging results, we proceeded to a phase I/IIa trial based upon repeated intra-arterial infusions of donor mesoangioblasts from HLA-matched siblings (Cossu et al. Embo Mol Med 2015). The trial was safe but showed little efficacy, mainly due to a very low engraftment of cells in the DMD muscles of patients selected, for safety reasons, at an advanced state of the disease. In those years, it became clear that cell therapy works well in those tissues such as epithelia and blood, where it is possible to remove diseased cells (surgically or by myeloablation) thus creating space for donor cells (Cossu et al. Lancet 2018). Since this strategy is not possible in tissues such as skeletal or cardiac muscle, then we have been developing different strategies to overcome the problem of low engraftment.

We can now enhance engraftment by tampering out steroids before cell transplantation: steroids reduce the binding of leukocytes but also circulating stem cells by inducing sialylation of VCAM and other endothelial receptors (Meggiolaro et al. under revision). However, the game changing result, recently published, is represented by a massive amplification of dystrophin production in DMD cells transduced with a 3^rd generation, self-inactivating lentiviral vector expressing, under the transcriptional control of the E1F- strong ubiquitous promoter, the U7 small nuclear RNA (U7 snRNA: De Angelis et al. PNAS 2002), engineered to skip exon 51 of the dystrophin gene. In other words, this is a “cell mediated exon skipping” where transduced cells act as “trojan horses”. Once fused with a regenerating muscle fibre, the donor cell will produce the U7 snRNA that will assemble with nuclear proteins and will enter not only the nucleus that produced it but also all neighbouring nuclei, thus amplifying of approximately one log dystrophin production (Galli et al. EMBO Mol Med 2024). Based on these results a phase I/IIa, supported by the Wellcome Trust, is now starting, as proof of principle, based upon a single intra-muscular injection of autologous, genetically corrected mesoangioblasts, in a single foot muscle, the Extensor Digitorum Brevis (EDB) of non-ambulant patients. After three months, dystrophin expression will be measured and if ³ 10% of a normal muscle, other cells will be injected in the thumb muscle whose force of contraction can be measured and, if increased, would benefit the hand function of non-ambulant patients. If successful, we will then move to systemic distribution, with an optimised protocol in very young patients, with “intent to cure”.

However, this personalized approach would prove prohibitively expensive for healthcare systems, as pricing of successful gene therapies is showing (De Luca & Cossu EMBO Rep. 2023). We made the striking observation that human mesoangioblasts can be indefinitely expanded with a novel culture medium, even after genetic manipulation and cloning. This now allows to perform experiments on huma adult progenitors that before were only possible in embryonic and reprogrammed stem cells, but without the risk of tumorigenesis and without chromosomal aberrations.

Therefore, donor mesoangioblasts have been first genome edited to delete endogenous HLA (b2-microglubin and class II CTA) while inserting tolerogenic HLA-E, fused to b2-microglubin and, as safety device, the CD20 suicide for negative selection as a safety devise. Edited clones will be checked for genome integrity and then tested for their ability to escape immune surveillance, first in vitro and then in vivo, in immune deficient DMD mice.

Engineered cells will be extensively characterised for safety and efficacy as ATIMP (Advanced Therapy Investigational Medicinal Product) and then stored in a master bank (to be later be a GMP grade master bank). From the master bank, cells will be engineered with specific tools to correct a specific mutation (cell-mediated exon skipping) in large genes such as dystrophin or dysferlin, or to replace a small gene whose full-length cDNA accommodates in a lentiviral vector.

The cell lines will be transplanted in humanized DMD mice and assessed for the ability to escape immune surveillance and to differentiate in myofibers expressing at high levels the healthy copy of the gene, thus establishing pre-clinical safety and efficacy for an off the shelf, affordable product. The group has unique expertise to successfully complete this project, whose strategy may be expanded to other recessive monogenic diseases, for a ground-breaking impact in regenerative medicine.