Immune-Privileged, immortal, myogenic stem cells for gene therapy of muscular dystrophy.
Duchenne muscular dystrophy (DMD) is a devastating incurable disease, affecting thousands with heavy burden on health systems. This project combines the development of a safe, “immune-privileged cell” with genetic engineering to correct many dystrophin gene mutations for an efficacious and cost affordable therapy. The applicant pioneered systemic intra-arterial transplantation of mesoangioblasts (blood vessel-derived progenitors) that proved safe in DMD patients and is being implemented for efficacy. However, this personalised approach would prove prohibitively expensive for healthcare systems, as pricing of successful gene therapies is showing. We made the striking observation that human mesoangioblasts can be indefinitely expanded with a novel culture medium, even after genetic manipulation and cloning. Cells will be first genome edited to delete endogenous HLA (β2-microglubin and class II CTA) while inserting tolerogenic HLA-E, fused to β2-microglubin and, as safety device, the Herpes Simplex Thymidine Kinase suicide gene with truncated NGF receptor for selection. Edited clones will be checked for genome integrity.  Selected clones will be engineered to express a small nuclear RNA (snRNA) that causes skipping of a given exon of the dystrophin gene. Due to the syncytial nature of muscle fibres, the snRNA also enters and corrects the genetic defect in neighbouring, dystrophic nuclei, thus amplifying of one log the therapeutic effect. Five different cell lines would correct the mutation in 60% of DMD patients.  The cell lines will be transplanted in humanized DMD mice and assessed for the ability to escape immune surveillance and to differentiate in dystrophin expressing myofibers, establishing pre-clinical safety and efficacy for an off the shelf, affordable product. The applicant 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.