Functional screening of expression libraries in vivo would offer the possibility of identifying novel biotherapeutics without a priori knowledge of their biochemical function. Here we describe a procedure for the functional selection of tissue-protective factors based on the in vivo delivery of arrayed cDNA libraries from the mouse secretome using adeno-associated virus (AAV) vectors. Application of this technique, which we call FunSel, in the context of acute ischaemia, revealed that the peptide ghrelin protects skeletal muscle and heart from ischaemic damage. When delivered to the heart using an AAV9 vector, ghrelin markedly reduces infarct size and preserves cardiac function over time. This protective activity associates with the capacity of ghrelin to sustain autophagy and remove dysfunctional mitochondria after myocardial infarction. Our findings describe an innovative tool to identify biological therapeutics and reveal a novel role of ghrelin as an inducer of myoprotective autophagy.
Progress in gene therapy has hinted at the potential misuse of gene transfer in sports to achieve better athletic performance, while escaping from traditional doping detection methods. Suitable animal models are therefore required in order to better define the potential effects and risks of gene doping. Here we describe a mouse model of gene doping based on adeno-associated virus (AAV)-mediated delivery of the insulin-like growth factor-I (IGF-I) cDNA to multiple muscles. This treatment determined marked muscle hypertrophy, neovascularization, and fast-to-slow fiber type transition, similar to endurance exercise. In functional terms, treated mice showed impressive endurance gain, as determined by an exhaustive swimming test. The proteomic profile of the transduced muscles at 15 and 30 days after gene delivery revealed induction of key proteins controlling energy metabolism. At the earlier time point, enzymes controlling glycogen mobilization and anaerobic glycolysis were induced, whereas they were later replaced by proteins required for aerobic metabolism, including enzymes related to the Krebs cycle and oxidative phosphorylation. These modifications coincided with the induction of several structural and contractile proteins, in agreement with the observed histological and functional changes. Collectively, these results give important insights into the biological response of muscles to continuous IGF-I expression in vivo and warn against the potential misuse of AAV-IGF1 as a doping agent.
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