Full‐length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells

Abstract

The protein dysferlin is abundantly expressed in skeletal and cardiac muscles, where its main function is membrane repair. Mutations in the dysferlin gene are involved in two autosomal recessive muscular dystrophies: Miyoshi myopathy and limb‐girdle muscular dystrophy type 2B. Development of effective therapies remains a great challenge. Strategies to repair the dysferlin gene by skipping mutated exons, using antisense oligonucleotides (AONs), may be suitable only for a subset of mutations, while cell and gene therapy can be extended to all mutations. AON‐treated blood‐derived CD133+ stem cells isolated from patients with Miyoshi myopathy led to partial dysferlin reconstitution in vitro but failed to express dysferlin after intramuscular transplantation into scid/blAJ dysferlin null mice. We thus extended these experiments producing the full‐length dysferlin mediated by a lentiviral vector in blood‐derived CD133+ stem cells isolated from the same patients. Transplantation of engineered blood‐derived CD133+ stem cells into scid/blAJ mice resulted in sufficient dysferlin expression to correct functional deficits in skeletal muscle membrane repair. Our data suggest for the first time that lentivirus‐mediated delivery of full‐length dysferlin in stem cells isolated from Miyoshi myopathy patients could represent an alternative therapeutic approach for treatment of dysferlinopathies.

Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle

“…44,45 In agreement with this assumption, engraftment and contribution to myofibers of nonmuscle stem cell types, like mesoangioblasts or CD133 + cells, have only been demonstrated in nonirradiated muscles of Scid/BLA/J mice, injured with myotoxins only, where the endogenous satellite cells were intact. 37,46 Our results clearly support the positive role of a double injury model. Although formation of functional satellite cells by transplanted myoblasts has been reported, 19,47 the detection of multiple Pax7-positive, dysferlin-positive cells in the graft's area was unexpected because engineered H2K A/J myoblasts had been extensively expanded ex vivo prior to transplantation.…”

“…37 In addition, there have been attempts to use RNA-based strategies like exon skipping or trans -splicing to treat dysferlinopathies. Exon 32 of DYSF might be dispensable for protein function, since a patient harboring a mutation in this exon developed a mild disease phenotype.…”

The Classification, Natural History and Treatment of the Limb Girdle Muscular Dystrophies

“…Another, non-subtype specific approach is to block myostatin, one of the body’s regulators of muscle growth [ 127 ]. Proteasome inhibition [ 128 ] and stem cell therapy are also under investigation [ 129 ]. With improved diagnostic tools, the identification of new drug targets and increased collaboration in the field or rare diseases, there is hope that more effective treatments will be developed for future use in patientswith LGMD.…”

Adult Stem Cells and Skeletal Muscle Regeneration

“…Recently, antisense oligonucleotide-based experiments have demonstrated a partial rescue of dysferlin levels in CD133 + stem cells isolated from patients with Miyoshi myopathy and subjected to myogenic induction. In addition, dysferlin-transduced CD133 + stem cells were able to affect positively the dystrophic phenotype of transplanted scid/blAJ dysferlin-null mice [147].…”