**9. Concluding remarks**

To date, many efforts have been made to increase the understanding of genetic and molecular mechanisms of different types of muscular dystrophy. Historically, the flux of knowledge achieved from patients to laboratories of research, or from bed to bench, allowed the design, test, production and administration of new molecules that only perform a partial restoration of dystrophin expression. Nonetheless, the use of common drugs, such as non-steroidal antiinflammatory drugs, corticosteroids and aminoglycosides antibiotics administered alone or combined with new drugs, showed poor beneficial effects regarding the restoration of dystrophin or the up-regulation of utrophin as compensatory mechanisms.

Thus, despite the limited results achieved by the pharmacological approaches tested so far, pharmacotherapy is still considered to be a useful tool in delaying the process of muscular degeneration and palliate the symptoms of the late stage of diseases.

An alternative approach for the treatment of dystrophies derives from the specific strategies adopted in gene therapy. Adeno-associated virus and lentiviral vector technologies have been studied in pre-clinical models to mediate the delivery of micro-dystrophin or mini-utrophin. Furthermore, an exon skipping strategy was proposed to restore the endogenous expression of dystrophin. Unfortunately, this also produced poor results in terms of dystrophin restora‐ tion and safety.

However, in recent decades, the stem cell therapy for muscular dystrophies has represented a new field of interest. In particular, it has sparked an increase in the understanding of biology in both multipotent (*i.e.* SP, MABs and FAPs) and pluripotent stem cells (*i.e.* ES and iPS cells), leading to the discovery and identification of new muscle progenitor cells. The exploitation of their myogenic potential has been investigated in several animal models including the mouse and dog. Extraordinary results were obtained in terms of dystrophin expression, decrease in inflammatory burden and increase in muscular function. More studies are likely to be conducted on myogenic derivatives from pluripotent stem cells and, possibly, in combination with miRNA and gene editing (CRISPR and TALEN) technologies for the treatment of muscle diseases. However, stem cell-based protocols still rely on adult stem cells. Many practical issues negatively interfere with their potential use in clinics. Low cell motility after transplan‐ tation, as well as the high immune rejection observed in pre-clinical models, are the main problems for obtaining a systemic treatment for cell-based therapy of dystrophies. A combined approach between pharmacotherapy and cell-based therapy can increase the beneficial effects of cell transplantation and give hope for the treatment of muscle degenerations. This could be achieved by increasing the myogenic differentiation potential of both multipotent and pluripotent stem cells by exosome, as well as miRNAs and gene editing technologies associated with pharmacological anti-inflammatory effects.

### **Acknowledgements**

Myostatin section). Our knowledge of miRNA biology is still in its infancy and future inves‐ tigation needs to be carried out in order to clarify the molecular mechanism and the precise

Recently, several groups tested gene editing to correct point mutation using TALEN and CRISPR genome editing. TALENs are endonucleases that possess two domains: a TAL effector DNA binding domain and a DNA cleavage domain. Left and right TALENs can induce a double strand break (DSB) in the DNA that allows homologous recombination of

Also, CRISPR (clustered regularly interspaced short palindromic repeats) is a RNA-guided gene-editing system that can introduce a double strand break at any desired location by delivering the Cas9 protein and appropriate guide RNAs. Olson group used CRISPR/Cas9 mediated genome editing to correct the dystrophin gene mutation in the germ line of mdx mice [56]. This procedure produced genetically mosaic animals, containing 2-100% correction of the dystrophin gene. In principle, this technology could facilitate the genome editing of postnatal somatic cells, avoiding the use of viral vectors. Li et al. recently performed three correc‐ tion methods (exon skipping, frame-shifting and exon knockin) in DMD-patient-derived iPSCs to restore the dystrophin protein. In their study, exon knockin was the most effective approach and identified clones with a minimal mutation load. We further investigated the genomic integrity by karyotyping, copy number variation array and exome sequencing to identify. TALEN and CRISPR-Cas9 corrected iPSCs were able to differentiate into skeletal muscle cells and express full-length dystrophin protein [57]. This is innovative technology that will be

To date, many efforts have been made to increase the understanding of genetic and molecular mechanisms of different types of muscular dystrophy. Historically, the flux of knowledge achieved from patients to laboratories of research, or from bed to bench, allowed the design, test, production and administration of new molecules that only perform a partial restoration of dystrophin expression. Nonetheless, the use of common drugs, such as non-steroidal antiinflammatory drugs, corticosteroids and aminoglycosides antibiotics administered alone or combined with new drugs, showed poor beneficial effects regarding the restoration of

Thus, despite the limited results achieved by the pharmacological approaches tested so far, pharmacotherapy is still considered to be a useful tool in delaying the process of muscular

An alternative approach for the treatment of dystrophies derives from the specific strategies adopted in gene therapy. Adeno-associated virus and lentiviral vector technologies have been studied in pre-clinical models to mediate the delivery of micro-dystrophin or mini-utrophin. Furthermore, an exon skipping strategy was proposed to restore the endogenous expression of dystrophin. Unfortunately, this also produced poor results in terms of dystrophin restora‐

involvement of these miRNAs in muscle development and regeneration.

further investigated. However, no field trials have been planned.

dystrophin or the up-regulation of utrophin as compensatory mechanisms.

degeneration and palliate the symptoms of the late stage of diseases.

the target DNA.

406 Muscle Cell and Tissue

**9. Concluding remarks**

tion and safety.

We would like to apologize to all authors whose work has not been reported here due to space limitations. This work has been supported by contributions from "Opening The Future" Campaign (EJJ-OPTFUT-02010), CARE-MI FP7, AFM Telethon, CARIPLO, FWO (#G060612N, #G0A8813N, #G088715N), GOA/11/012, IUAP-VII/07 and OT#09-053 grants. EB is supported by an FWO Post-doctoral Fellowship (12D2813N) and an FWO grant (1525315N). We thank Christina Vochten and Vicky Raets for their professional administrative assistance. We would also like to thank Rondoufonds voor Duchenne Onderzoek for their kind donations. PGD

### **Author details**

Emanuele Berardi1 and Maurilio Sampaolesi1,2\*

\*Address all correspondence to: maurilio.sampaolesi@med.kuleuven.be

1 Translational Cardiomyology Laboratory, Stem Cell Biology and Embryology Unit, De‐ partment of Development and Regeneration, KU Leuven, Belgium

2 Division of Human Anatomy, Department of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy
