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Improved Gene Correction Strategy for Muscular Dystrophy Therapeutics



Review of “Ex vivo gene editing of the dystrophin gene in muscle stem cells using peptide nucleic acid single stranded oligodeoxynucleotides (PNA-SsODNs) induces stable expression of dystrophin in a mouse model for Duchenne muscular dystrophy” from Stem Cells by Stuart P. Atkinson.

One of the beneficial therapeutic approaches for Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene leading to the complete loss of the protein [1-3], is to restore shorter, but still functional, dystrophin protein. Single‐stranded oligodeoxynucleotide (ssODN) expression in muscle cells are able to induce single base‐pair alterations to correct mutations [4]; however ssODNs composed of peptide nucleic acid bases (PNA‐ssODNs) are capable of higher frequency gene correction [5]. A study published in Stem Cells from Nik‐Ahd and Bertoni at the Department of Neurology, David Geffen School of Medicine, University of California, USA, have now shown the utility of PNA‐ssODNs for gene repair in muscle stem cells (or satellite cells – SCs [6]) isolated from a mouse model of DMD, and the generation of new myofibers, fusion with pre-existing fibers, and reparation of damaged fibers [7].

The researchers first purified SCs from the muscle of DMD mice which, in comparison to WT mice, displayed a significantly higher percentage of cells expressing MyoD, desmin and myogenin likely due to the response of dystrophin-negative muscles activating SCs to replace myofibers that have degenerated. Time course transfection analysis of labelled PNA‐ssODNs found a 95% uptake efficiency at 24 hours, which declined between 72 and 96 hours. Gene repair assessment at 2 weeks revealed expression of dystrophin in some of the cultures propagated in vitro which were then induced to differentiate for 48 hrs, although the level of full‐length dystrophin protein only ranged between 1% and 2% of that of wild‐type cells. Genomic DNA analyses confirmed the presence of corrected dystrophin DNA, with the efficiency of frequency of repair (1.5% to 2.1%) correlating to previous results.

The authors then assessed the ability of SCs, transfected with PNA‐ssODN as described above, to restore dystrophin expression in the DMD mouse model. The authors detected dystrophin+ fibers in all tibialis‐anterior (TA) muscles that received corrected SCs, while injury to TA muscles in engrafted mice also led to an increase in the number of dystrophin+ fibers, as expected for wild type mice. Using immunostaining of Pax7 and MyoD expression, markers of quiescent and activated SCs respectively, the researchers detected Pax7‐positive cells in both muscles of control mice and muscles of DMD mice engrafted with corrected SCs, suggesting that engrafted cells had retained the ability to divide and to give rise to muscle progenitor cells. The longitudinal distribution of dystrophin in injured muscle following corrected SC transplantation (and wild-type SCs) was also greater than that detected in resting muscles, while the number of fibers in each cluster expressing dystrophin was one to two‐ fold higher than that observed in non-injured SC receiving muscles. This altogether suggested that engrafted corrected SCs could return to quiescence, were able to exit the quiescent state when prompted, and could contribute to muscle regeneration.

Long term analysis (at 24 weeks) found large clusters of full length dystrophin+ fibers in muscles transplanted with both corrected and wild type SCs. An increase in dystrophin+ fiber number between 5 and 24 weeks suggested that, as before, repaired SCs could contribute to muscle regeneration. The lack of differences in the number of quiescence and activated SCs in muscles isolated 24 weeks after SC transplantation suggested that corrected SCs function was not due to differences in engraftment or survival of cells over time, and was more likely due to an increase in the number and distribution of dystrophin‐positive fibers. Analysis of myonuclei localization in dystrophin‐positive fibers was then undertaken and used as an index of functional recovery [8] following SC transplantation. Upon corrected SC or wild type SC transplant, dystrophin‐positive fibers displayed central nuclei demonstrating regeneration and/or repair of those fibres, although this effect became decreased by 24 weeks, and was further associated with a decreased level of dystrophin expression within individual fibers.

This excellent study suggests that ssODN‐mediated gene repair in SCs leads to the recovery of functional dystrophin expression in individual muscle fibers to a level which is therapeutically relevant, placing this strategy at the forefront for the treatment of DMD and indeed other neuromuscular diseases. Gene repair in these SCs took place in the minority of cells, but these results provide strong evidence that even this low level of correction is sufficient to achieve sustained beneficial effects. The authors note that further studies are required to address safety concerns surrounding the gene correction, and to further characterize SCs that have undergone gene repair to develop new strategies to improving targeting efficiencies of ssODNs, and that they are currently assessing methods to use the circulatory system as the method of delivering stem cells to achieve maximal muscle targeting, and also testing the feasibility of delivering PNA‐ssODNs into muscles after systemic administration to target SCs in vivo.


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