Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice.

Background: Gene doping is the misuse of genome editing and gene therapy technologies for the purpose of manipulating specific genes or gene functions in order to improve athletic performance. However, a non-invasive detection method for gene doping using recombinant adenoviral (rAdV) vectors containing human follistatin () genes (rAdV<>) has not yet been developed. Therefore, the aim of this study was to develop a method to detect gene doping using rAdV<>.

Methods: First, we generated rAdV<> and evaluated the overexpression of the gene, FST protein, and muscle protein synthesis signaling using cell lines. Next, rAdV<> was injected intravenously or intramuscularly into mice, and whole blood was collected, and and cytomegalovirus promoter () gene fragments were detected using TaqMan-quantitative polymerase chain reaction (qPCR). Finally, to confirm the specificity of the primers and the TaqMan probes, samples from each experiment were pooled, amplified using TaqMan-qPCR, and sequenced using the Sanger sequencing.

Results: The expression of hFST and FST proteins and muscle protein synthesis signaling significantly increased in C2C12 cells. In long-term, transgene fragments could be detected until 4 days after intravenous injection and 3 days after intramuscular injection. Finally, the Sanger sequencing confirmed that the primers and TaqMan probe specifically amplified the gene sequence of interest.

Conclusions: These results indicate the possibility of detecting gene doping using rAdV<> using TaqMan-qPCR in blood samples. This study may contribute to the development of detection methods for gene doping using rAdV<>.