**8. New frontiers for the treatment of MDs: Exosomes, MicroRNAs and gene editing**

MicroRNAs (miRs) are non-coding RNA transcripts, ∼22 nucleotides long that promote mRNA degradation by annealing to complementary sequences in the 3'untranslated regions (UTR) of specific target mRNA. Furthermore, miRs can target several transcripts and system individual mRNAs can be targeted by multiple miRs.

The biogenesis of miRNAs starts with the generation of pri-miRs by RNA polymerase II. These pri-miRs are transformed in pre-miRs by the microprocessor complex. They then transport them in the cytosol, where Dicer cleaves pre-miRs in ~22nt-long double-stranded molecules [50]. The guide strand responsible for the recognition of target mRNAs is loaded on the RNAinduced silencing complex (RISC), which contains multiple proteins including a ribonuclease enzyme.

Several biological processes, including muscle growth and differentiation, are mediated by a collection of specific miRs. These miRs can be released from the cells in the surrounding areas or in the circulation and circulating miRs (circ-miRs) appear resistant to harsh conditions [51]. Circ-miRs are protected by carriers, making them stable and valuable biological markers. Among the different carriers identified, exosomes are small vesicles (50-100nm diameter) that act as important regulators of long-range miR shuttling [52]. After the unknown processes of maturation, exosomes are released from the plasma membrane and are identified by specific markers, as Hsp-60/70 in the lumen and CD9/63/81 and tissue-specific membrane proteins on the surface [53]. Despite the lack of detail concerning receptors and intracellular processing generate debate and controversies, it is largely accepted that pre- or mature miRs are delivered to other cells, eliciting their regulation in target non-miR-originating cells.

The importance of miRNAs in the muscle development was established in a study involving conditional transgenic mice lacking Dicer in myogenic progenitors. This study resulted in aberrant muscle differentiation, accompanied by hyperplasia [54].

Furthermore, miR-206 is the most abundant miRNA in adult vertebrate skeletal muscle and promotes muscle skeletal muscle development and differentiation [55].

Interestingly, a mutation in myostatin gene that causes a dramatic muscle increase in textil sheep creates a target site for miR-206 and miRNA1. In these sheep, myostatin down-regulation determines a phenocopy of the double muscling Belgiun Blue cattle previously described (see 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 involvement of these miRNAs in muscle development and regeneration.

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 the target DNA.

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