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Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping

Gene Therapy volume 18, pages 709–718 (2011)Cite this article

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Abstract

Legitimate uses of gene transfer technology can benefit from sensitive detection methods to determine vector biodistribution in pre-clinical studies and in human clinical trials, and similar methods can detect illegitimate gene transfer to provide sports-governing bodies with the ability to maintain fairness. Real-time PCR assays were developed to detect a performance-enhancing transgene (erythropoietin, EPO) and backbone sequences in the presence of endogenous cellular sequences. In addition to developing real-time PCR assays, the steps involved in DNA extraction, storage and transport were investigated. By real-time PCR, the vector transgene is distinguishable from the genomic DNA sequence because of the absence of introns, and the vector backbone can be identified by heterologous gene expression control elements. After performance of the assays was optimized, cynomolgus macaques received a single dose by intramuscular (IM) injection of plasmid DNA, a recombinant adeno-associated viral vector serotype 1 (rAAV1) or a rAAV8 vector expressing cynomolgus macaque EPO. Macaques received a high plasmid dose intended to achieve a significant, but not life-threatening, increase in hematocrit. rAAV vectors were used at low doses to achieve a small increase in hematocrit and to determine the limit of sensitivity for detecting rAAV sequences by single-step PCR. DNA extracted from white blood cells (WBCs) was tested to determine whether WBCs can be collaterally transfected by plasmid or transduced by rAAV vectors in this context, and can be used as a surrogate marker for gene doping. We demonstrate that IM injection of a conventional plasmid and rAAV vectors results in the presence of DNA that can be detected at high levels in blood before rapid elimination, and that rAAV genomes can persist for several months in WBCs.

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Acknowledgements

We thank Michael L Nickerson, PhD (NCI Frederick), Mahajoub Bello-Roufai, PhD (UF CERHB), Oumeya Adjali, MD, PhD, James Baus for helpful discussions, Yan Chérel, DVM, PhD, Béatrice Joussemet, DVM, PhD, and Delphine Nivard for technical assistance, the personnel at the Boisbonne Centre (large animal facility, ONIRIS, Nantes) and the Vector Core at the University Hospital of Nantes for providing the rAAV1 and rAAV8 stocks. This project was funded by the World Anti-Doping Agency (ROS and PM), United States Anti-Doping Agency (ROS) and the Agence Francaise de Lutte contre le Dopage (PM). This work was performed under a Cooperative Agreement between INSERM, AFM, l’Etablissement Francais du Sang (EFS) and the University of Florida Center of Excellence for Regenerative Health Biotechnology.

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  1. W Ni and C Le Guiner: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL, USA

    W Ni, M Penaud-Budloo, P Moullier & R O Snyder

  2. Laboratoire de Thérapie Génique, INSERM UMR649, IRT UN, Nantes, France

    C Le Guiner, G Gernoux, M Penaud-Budloo, P Moullier & R O Snyder

  3. GENETHON, Evry, France

    C Le Guiner & P Moullier

  4. Department of Pediatrics, College of Medicine, University of Florida, FL, USA

    R O Snyder

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  1. W Ni
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Correspondence to R O Snyder.

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Competing interests

ROS is an inventor on patents related to recombinant AAV technology. ROS owns equity in a gene therapy company that is commercializing AAV for gene therapy applications. To the extent that the work in this manuscript increases the value of these commercial holdings, ROS has a conflict of interest.

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Ni, W., Le Guiner, C., Gernoux, G. et al. Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping. Gene Ther 18, 709–718 (2011). https://doi.org/10.1038/gt.2011.19

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