Proof of Gene Doping in a Mouse Model with a Human Erythropoietin Gene Transferred Using an Adenoviral Vector

Despite the World Anti-Doping Agency (WADA) ban on gene doping in the context of advancements in gene therapy, the risk of EPO gene-based doping among athletes is still present. To address this and similar risks, gene-doping tests are being developed in doping control laboratories worldwide. In this regard, the present study was performed with two objectives: to develop a robust gene-doping mouse model with the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to identify gene doping by using this model. The rAdV including the hEPO gene was injected intravenously to transfer the gene to the liver. After injection, the mice showed significantly increased whole-blood red blood cell counts and increased expression of hematopoietic marker genes in the spleen, indicating successful development of the gene-doping model. Next, direct and potentially indirect proof of gene doping were evaluated in whole-blood DNA and RNA by using a quantitative PCR assay and RNA sequencing. Proof of doping could be detected in DNA and RNA samples from one drop of whole blood for approximately a month; furthermore, the overall RNA expression profiles showed significant changes, allowing advanced detection of hEPO gene doping.