Establishment of two quantitative nested qPCR assays targeting the human EPO transgene

Abstract

For ethical and safety reasons it is critical to develop easily implemented assays with high sensitivity and specificity for gene doping surveillance. Two nested quantitative real-time PCR (qPCR) assays were developed that target the human EPO (hEPO) cDNA sequence in a circular form, representative of recombinant adeno-associated viral (rAAV) vector genomes found in vivo. Through an interlaboratory evaluation, the assays were validated and utilized in an in vitro blinded study. These assays are specific and extremely sensitive with a limit of detection (LOD) of 1 copy of circular plasmid DNA and a limit of quantification (LOQ) of 10 to 20 copies in the presence of 500 ng of human genomic DNA (hgDNA) extracted from WBCs. Additionally, using the two nested qPCR assays in a non-human primate study, where macaques were injected intramuscularly with a rAAV8 vector harboring a promoterless hEPO cDNA sequence, the viral vector was detected 8 to 14 weeks post-injection in macaque WBCs. The high sensitivity of the nested qPCR approach along with the capability of quantifying target DNA, make this approach a reliable tool for gene doping surveillance and the monitoring of exogenous DNA sequences.

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Acknowledgements

We thank Aude Morvan and Sophie Moullec for their technical assistance during the IM injections in macaques. We also thank all the staff from the Boisbonne Center for the handling and care of the macaques. Moreover, we thank the Vector Core of Atlantic Gene Therapies, Hospital of Nantes (Nantes, France) for providing the rAAV8-promorterless hEPO cDNA vector and Achille Francois for helpful discussion. We also acknowledge WADA 11C9RS (ROS, PS, PM), PCC (ROS), and the NSF Training Grant #DGE1011553 (ROS) for their funding and support. We also would like to thank Johanne Le Duff for her help in the immunological analysis of the serum from the macaques, and Linda Orsini, Belinda Manukian, and Dr. Craig Fletcher from the University of Florida Blood Bank. Furthermore, this study was conducted under a Cooperative Agreement between Institut National de la Sante et de la Recherche Medicale, Association Francaise Contre Les Myopathies, l'Etablissement Francais du Sang and the University of Florida Center of Excellence for Regenerative Health Biotechnology.

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Correspondence to E W I Neuberger.

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ROS is an inventor on patents related to rAAV technology. ROS owns equity in a gene therapy company that is commercializing adeno-associated virus for gene therapy applications. To the extent that the work reported in this article increases the value of these commercial holdings, R.O.S. has a conflict of interest.

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Neuberger, E., Perez, I., Le Guiner, C. et al. Establishment of two quantitative nested qPCR assays targeting the human EPO transgene. Gene Ther 23, 330–339 (2016). https://doi.org/10.1038/gt.2016.2

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