A Gene Therapy Approach for Treating Muscle Degeneration in Neuromuscular Disorders Using IGF-I Splice Variants

N eurom uscular disorders, which associate with muscle degeneration, are not rare and affect millions o f people worldwide. The impacts o f such disorders vary from gradual loss o f mobility and independence to severe disability and death, and therefore m illions o f patients suffer from them at every stage o f their life. Because, there is currently no treatment o f any form o f such disorders, this study was aimed to develop a novel treatm ent for such disorders. For a long time it has been known that Insulin-like Growth Factor (IGF-I) influences several cellular processes, including proliferation, differentiation, repair and maintenance. Like m any genes, the IGF-I gene can be spliced to produce several isoforms, and in hum an muscle, it expresses at least two main isoforms which are a liver type, systemic form (IGF-I Ea) and an autocrine / paracrine form (IGF-I Ec). This second isoform has been named the Mechano Growth Factor (MGF) because o f its mechanosensitivity. The in vitro and in vivo effects o f these two splice variants o f the IGF-I gene were investigated in this study. In vitro roles o f two splice variants o f the gene were studied by the proliferation / differentiation effects o f two alternative splice isoforms o f the gene in animal muscle cell lines (mouse C2C12 and rat L6 E9). Proliferation / differentiation assays were carried out using human primary cell cultures from biopsied muscles from congenital muscular dystrophy (CMD), fascioscapulohumeral m uscular dystrophy (FSHD) and amyotrophic lateral sclerosis (ALS) patients as well as from healthy volunteers. Human primary muscle cells were treated with IGF-I Ea (long r IGF-I) and MGF E domain peptides, and immunocytochemistry techniques with Desmin, DAPI and FITC markers were used to detect proliferation state o f myogenic commitment. The CPK and BCA protein assays were also used to determ ine the differentiation state following such peptide treatments. The results showed that MGF significantly increased muscle stem (satellite) cell proliferation in both animal and human muscles, both in healthy and in severe muscle wasting disorders. E dom ain o f MGF dramatically increased proliferation in progenitor cell in CMD (68%), FSHD (74% ) and ALS (49%) primary cultures. The results also confirmed that the M GF had no effect on myotube formation but that it increases myoblast progenitor cell proliferation, whilst systemic IGF-I peptide (IGF-I Ea) increased cell differentiation and facilitated myotube formation. The effects o f two IGF-I splice variants in muscle fibre growth were also studied in relation to Duchenne M uscular Dystrophy (DMD) by in vivo gene transfer method using the mdx mouse model. Such effects were investigated in both young and old mdx mice TA muscles by intramuscular injection o f cDNAs in plasmid vectors pcD NA 3.1NT/GFP. Maximum muscle tetanic contractile force was measured to determine the changes o f muscle strength at 21 days after gene injection. The results showed that cDNA o f MGF dramatically increased muscle fibre strength in young mdx mice (37 %) in only 3 weeks time. The MGF also increased muscle strength and