Transplantation of genetically corrected Duchenne muscular dystrophy (DMD) patient-derived induced pluripotent stem cells (DMD-iPSCs) could represent a potential therapeutic alternative for patients with DMD, according to a recently published study in the International Journal of Molecular Sciences.
Currently, patients with DMD are mostly treated with corticosteroids, which delay muscle atrophy but do not prevent it. Thus, there is a growing interest in developing genetic therapies with the potential to improve quality of life and prolong the survival of these patients. However, the large size of the DMD gene makes exogenous gene delivery and addition a challenge.
Ex vivo gene therapy is a promising alternative for DMD. Due to their proliferation capacity and multidirectional differentiation potential, DMD-iPSCs represent a promising cell source for performing homologous-directed recombination-based gene correcting and autologous transplantation.
The authors aimed to restore dystrophin expression in DMD-iPSCs by adding the missing exon via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. The authors hypothesized that the genetically corrected cells could differentiate into fully functional cardiomyocytes.
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Researchers used urinary cells with a deleted exon 50 from a patient with DMD to generate a line of DMD-iPSCs. Afterward, 2 single-guide RNAs were used to insert exon 50 in situ. The genomic DNA of normal human iPSCs (hiPSCs) was the template used to amplify exon 50 of DMD and its splicing donor sequence.
Forty-eight hours after conucleofection of CRISPR/Cas9 and the donor template plasmid, the authors performed G418 drug screening for 3 days. Survivor cells were later digested into single cells for monoclonal formation, producing 15 single clones. After performing agarose gel electrophoresis and Sanger sequencing, only 1 clone (Rn14-iPSCs) was selected.
Posterior sequencing revealed that Rn14-iPSCs were identical to hiPSCs and that exon 50 was, in effect, inserted into DMD in Rn14-iPSCs, thus restoring its splicing and transcription. Rn14-iPSCs retained pluripotency marker expression. Teratomas derived from Rn14-iPSCs revealed that Rn14-iPSCs could differentiate into all 3 germ layers.
The genetically corrected Rn14-iPSCs were differentiated into cardiomyocytes with a small molecule-induced protocol. The resulting cardiomyocytes expressed troponin T and were able to contract spontaneously.
“This is the first study demonstrating that full-length dystrophin can be restored in iPSCs and [iPSC-derived cardiomyocytes] via targeted exon addition, indicating potential clinical prospects for DMD gene therapy,” the authors concluded.
Xiao R, Zhou M, Wang P, et al. Full-length dystrophin restoration via targeted exon addition in DMD-patient specific iPSCs and cardiomyocytes. Int J Mol Sci. 2022;23(16):9176. doi:10.3390/ijms23169176