Gene therapy for Duchenne muscular dystrophy: an update on the latest clinical developments.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is one of the most severe and devastating neuromuscular hereditary diseases with a male newborn incidence of 20 000 cases each year. The disease caused by mutations (exon deletions, nonsense mutations, intra-exonic insertions or deletions, exon duplications, splice site defects, and deep intronic mutations) in the DMD gene, progressively leads to muscle wasting and loss of ambulation. This situation is painful for both patients and their families, calling for an emergent need for effective treatments. AREAS COVERED: In this review, the authors describe the state of the gene therapy approach in clinical trials for DMD. This therapeutics included gene replacement, gene substitution, RNA-based therapeutics, readthrough mutation, and the CRISPR approach. EXPERT OPINION: Only a few drug candidates have yet been granted conditional approval for the treatment of DMD. Most of these therapies have only a modest capability to restore the dystrophin or improve muscle function, suggesting an important unmet need in the development of DMD therapeutics. Complementary genes and cellular therapeutics need to be explored to both restore dystrophin, improve muscle function, and efficiently reconstitute the muscle fibers in the advanced stage of the disease.

Prime editing strategies to mediate exon skipping in DMD gene.

Duchenne muscular dystrophy is a rare and lethal hereditary disease responsible for progressive muscle wasting due to mutations in the DMD gene. We used the CRISPR-Cas9 Prime editing technology to develop different strategies to correct frameshift mutations in DMD gene carrying the deletion of exon 52 or exons 45 to 52. With optimized epegRNAs, we were able to induce the specific substitution of the GT nucleotides of the splice donor site of exon 53 in up to 32% of HEK293T cells and 28% of patient myoblasts. We also achieved up to 44% and 29% deletion of the G nucleotide of the GT splice site of exon 53, as well as inserted 17% and 5.5% GGG between the GT splice donor site of exon 51 in HEK293T cells and human myoblasts, respectively. The modification of the splice donor site for exon 51 and exon 53 provoke their skipping and allowed exon 50 to connect to exon 53 and allowed exon 44 to connect to exon 54, respectively. These corrections restored the expression of dystrophin as demonstrated by western blot. Thus, Prime editing was used to induce specific substitutions, insertions and deletions in the splice donor sites for exons 51 and 53 to correct the frameshift mutations in DMD gene carrying deletions of exon 52 and exons 45 to 52, respectively.

Prime editing optimized RTT permits the correction of the c.8713C>T mutation in DMD gene.

Duchenne muscular dystrophy is a severe debilitating genetic disease caused by different mutations in the DMD gene leading to the absence of dystrophin protein under the sarcolemma. We used CRISPR-Cas9 prime editing technology for correction of the c.8713C>T mutation in the DMD gene and tested different variations of reverse transcription template (RTT) sequences. We increased by 3.8-fold the editing percentage of the target nucleotide located at +13. A modification of the protospacer adjacent motif sequence (located at +6) and a silent mutation (located at +9) were also simultaneously added to the target sequence modification. We observed significant differences in editing efficiency in interconversion of different nucleotides and the distance between the target, the nicking site, and the additional mutations. We achieved 22% modifications in myoblasts of a DMD patient, which led to dystrophin expression detected by western blot in the myotubes that they formed. RTT optimization permitted us to improve the prime editing of a point mutation located at +13 nucleotides from the nick site to restore dystrophin protein.

Improvements of nuclease and nickase gene modification techniques for the treatment of genetic diseases.

Advancements in genome editing make possible to exploit the functions of enzymes for efficient DNA modifications with tremendous potential to treat human genetic diseases. Several nuclease genome editing strategies including Meganucleases (MNs), Zinc Finger Nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas) have been developed for the correction of genetic mutations. CRISPR-Cas has further been engineered to create nickase genome editing tools including Base editors and Prime editors with much precision and efficacy. In this review, we summarized recent improvements in nuclease and nickase genome editing approaches for the treatment of genetic diseases. We also highlighted some limitations for the translation of these approaches into clinical applications.

Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular Dystrophy.

The Prime editing technique derived from the CRISPR/Cas9 discovery permits the modification of selected nucleotides in a specific gene. We used it to insert specific point mutations in exons 9, 20, 35, 43, 55 and 61 of the Duchenne Muscular Dystrophy (DMD) gene coding for the dystrophin protein, which is absent in DMD patients. Up to 11% and 21% desired mutations of the DMD gene in HEK293T cells were obtained with the PRIME Editor 2 (PE2) and PE3, respectively. Three repeated treatments increased the percentage of specific mutations with PE2 to 16%. An additional mutation in the protospacer adjacent motif (PAM) sequence improved the PE3 result to 38% after a single treatment. We also carried out the correction of c.428 G>A point mutation in exon 6 of the DMD gene in a patient myoblast. Myoblast electroporation showed up to 8% and 28% modifications, respectively, for one and three repeated treatments using the PE3 system. The myoblast correction led to dystrophin expression in myotubes detected by Western blot. Thus, prime editing can be used for the correction of point mutations in the DMD gene.

Therapeutic Strategies for Dystrophin Replacement in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked hereditary disease characterized by progressive muscle wasting due to modifications in the DMD gene (exon deletions, nonsense mutations, intra-exonic insertions or deletions, exon duplications, splice site defects, and deep intronic mutations) that result in a lack of functional dystrophin expression. Many therapeutic approaches have so far been attempted to induce dystrophin expression and improve the patient phenotype. In this manuscript, we describe the relevant updates for some therapeutic strategies for DMD aiming to restore dystrophin expression. We also present and analyze in vitro and in vivo ongoing experimental approaches to treat the disease.

CRISPR-Cas9 Gene Therapy for Duchenne Muscular Dystrophy.

Discovery of the CRISPR-Cas (clustered regularly interspaced short palindromic repeat, CRISPR-associated) system a decade ago has opened new possibilities in the field of precision medicine. CRISPR-Cas was initially identified in bacteria and archaea to play a protective role against foreign genetic elements during viral infections. The application of this technique for the correction of different mutations found in the Duchenne muscular dystrophy (DMD) gene led to the development of several potential therapeutic approaches for DMD patients. The mutations responsible for Duchenne muscular dystrophy mainly include exon deletions (70% of patients) and point mutations (about 30% of patients). The CRISPR-Cas 9 technology is becoming increasingly precise and is acquiring diverse functions through novel innovations such as base editing and prime editing. However, questions remain about its translation to the clinic. Current research addressing off-target editing, efficient muscle-specific delivery, immune response to nucleases, and vector challenges may eventually lead to the clinical use of the CRISPR-Cas9 technology. In this review, we present recent CRISPR-Cas9 strategies to restore dystrophin expression in vitro and in animal models of DMD.