Molecular signatures of IGF-1 gene doping after AAV-mediated gene transfer

Code: 06B06MG

This is a highly interdisciplinary and coordinated project that is aimed at understanding the molecular modifications induced by prolonged IGF-1 gene expression in the skeletal muscle after viral mediated gene transfer in rodent animals. The project will exploit the availability of AAV vectors expressing different IGF-1 isoforms to transduce skeletal muscles, a unique system that permites the long-term evaluation of the effects induced by the growth factor in vivo. In the treated animals, the signatures of these modifications will be analyzed by detecting the presence of the delivered transgenes in serum by quantitative real-time PCR and by analyzing the modifications of the proteomic pattern in muscle by advanced proteomics and mass spectrometry. These studies will be complemented by a parallel evaluation of the proteomic modifications induced by IGF-1 gene transfer in skeletal muscle satellite cells in vitro.

The project will be divided into three tasks.

Task 1. The overall purpose of this task will be the identification of a proteomic signature of IGF-1 gene transfer in muscle cells. The project will involve the development of animal models for AAV-mediated IGF-1 gene transfer. In particular, viral vectors expressing different IGF-1 isoforms will be produced and used to inoculate the tibialis anterior and femoral quadriceps muscles of mice and rats. At different times after transduction, proteomic analysis of the transduced muscles will be performed. These studies will take advantage of state-of-the-art technology 2-DE DIGE and will have the ultimate purpose of identifying protein patterns specific to the IGF-1 expressing muscles. These studies will be paralleled by the analysis of proteomic changes in human skeletal myoblasts after ex vivo gene transfer of the IGF-1 cDNAs.

Task 2. This task is aimed at the identification of novel peptides markers that might be exploited for anti-doping purposes. In particular, the project is aimed at the indentifications, quantification and qualitative assessment of the proteins differential expressed in muscle transduced with IGF-1. Differentially expressed or modified proteins will be analyzed using advanced mass spectrometry instrumentation, including MALDI-TOF/TOF, ESI-linear quarupole ion trap and high resolution FT-MS instrumentation.

Task 3. This task is aimed at the assessment of gene doping by monitoring the presence of exogenous gene fragments in animal serum. This possibility is based on different experimental and clinical reports that indicate that muscle exercise determines signficant damage to muscle cells, with the release of intracellular content into the bloodstream. At different times after in vivo IGF-1 gene transfer, both in resting conditions and during muscle exercise, serum will be analyzed for the presence of promoter-, vectore backbone- or cDNA-specific gene fragments by TaqMan-based Real Time quantitative PCR.

Main findings

The main findings are not available due to the sensitivity of the information and results developed in this project.