In recent years, as gene therapy technology has rapidly developed, there has been growing concern that it could be misused by athletes as a means of doping. However, current testing methods for gene doping have a range of limitations and require further improvement. Furthermore, significant progress has been made in the fields of blood storage, next-generation sequencing (NGS), and LabDroid (experimental robots). Against this background, this study was implemented to develop a test method for gene doping using dried blood spot (DBS), NGS, and the LabDroid ”Maholo”. As a first step, recombinant adeno-associated virus containing the human erythropoietin gene (hEPO) was injected into mice to establish a gene doping model. Subsequently, DBS was created using whole blood. Maholo was used to extract DNA from the DBS and to create DNA libraries for NGS. NGS in combination with bioinformatic analysis clearly identified DNA fragments that provided definitive evidence of gene doping in the mouse model, which were absent in the control mouse. To the best of our knowledge, this is the first attempt to use a biological model of hEPO gene doping in conjunction with Maholo, NGS, and DBS. This method should facilitate the further development of gene doping tests.

Regular bathing is a common habit worldwide, yet bath additives have been characterized almost exclusively at the physiological level, while their molecular effects on human cells remain largely unexplored. We previously showed by RNA sequencing (RNA-seq) that the complex bath additive Karada Totonou ProBath (KTPB) induced the expression of EGR1 and hyaluronic acid synthase genes in human keratinocytes and fibroblasts, which represent the vascular endothelial growth factor (VEGF)-producing side of the cutaneous angiogenic axis; whether the VEGF-receiving endothelium responds to KTPB was unknown. Here, human vascular endothelial cells were exposed to KTPB and profiled by RNA-seq, and cytotoxicity was assessed using resazurin and Hoechst assays. KTPB altered gene expression in a time-dependent manner, and the differentially expressed genes were classified into three clusters with distinct temporal profiles: a late-repressed cluster, a transiently induced cell cycle-associated cluster, and a late-induced cluster enriched for blood vessel development and VEGFA-VEGFR2 signaling. The angiogenesis-related genes ID1, ID3, and EDN1 were markedly upregulated, peaking at 1–2 h. KTPB showed no detectable cytotoxicity in this cell model at any of the tested concentrations, which spanned the recommended use range. These results indicate that KTPB elicits an angiogenesis-associated transcriptional program in vascular endothelial cells without compromising viability and highlight transcriptomics’ value for characterizing bath additives.