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

Abstract

The World Anti-Doping Agency (WADA) has prohibited gene doping in the context of progress in gene therapy. In addition, there is a risk of the EPO gene being applied in gene doping among athletes. Along with this, development of a gene-doping test has been underway in worldwide. Here, we had two purposes: to develop a robust gene doping mouse model using the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to prove gene doping using this model. The rAdV including the hEPO gene were injected intravenously to transfer the gene to the liver. After injection, the mice developed significantly increased red blood cell counts in whole blood and increased gene expressions of hematopoietic markers in the spleen, indicating successful development of the gene doping model. Next, we detected direct and indirect proof of gene doping in whole blood DNA and RNA using qPCR assay and RNA sequencing. Proof was detected in one drop of whole blood DNA and RNA over a long period; furthermore, the overall RNA expression profiles significantly changed. Therefore, we have advanced detection of hEPO gene doping in humans.

Annual banned‐substance review: Analytical approaches in human sports drug testing 2020/2021

“…In January 2021, WADA released laboratory guidelines regarding gene doping detection methods employing polymerase chain reaction (PCR), 150 and several studies elaborating on currently available models and analytical approaches for detecting transgenes for sports drug testing purposes as well as strengths and limitations were published 112 . For instance, Sugasawa et al 113 reported on the development of a mouse model transfected with a human EPO gene through an adenoviral vector, which allowed for assessing options of direct (transgene‐focused) and indirect (longitudinal monitoring of RNA expressions) anti‐doping testing strategies. Mice subjected to injections with human EPO‐gene‐containing adenoviral vectors (rAdV‐hEPO) showed significantly increased red blood cell counts and elevated hematopoietic marker gene expressions, indicating the successful gene transfer.…”

“…109 An important aspect of the applicability of the hematological ABP (PCR), 150 and several studies elaborating on currently available models and analytical approaches for detecting transgenes for sports drug testing purposes as well as strengths and limitations were published. 112 For instance, Sugasawa et al 113 reported on the development of a mouse model transfected with a human EPO gene through an adenoviral vector, which allowed for assessing options of direct…”