Raising the bar for gene therapy tests

Researchers develop a strategy to improve contamination detection in gene-doping tests.

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A team of researchers in Australia has developed a new strategy to design synthetic reference materials for tests that detect gene doping or the illegal use of gene therapy by athletes. 

Unscrupulous athletes could potentially use gene therapy to enhance performance by tweaking their genes or introducing additional copies of genes, such as EPO, which regulates red blood cell production.While gene doping remains a hypothetical problem, the World Anti-Doping Agency has been funding research on detection techniques since 2004.  

Gene doping tests routinely include a ‘positive’ sample that contains the genetic material being tested for in order to verify that the detection procedure is working properly.However, DNA from the positive control may contaminate the test sample, resulting in a false result. Even mild contamination can prove problematic since most of the tests involve the amplification of minute quantities of DNA. 

Anna Baoutina of Australia’s National Measurement Institute and her colleagues developed a reference design strategy that overcomes the problem of cross-contamination. To detect the foreign genetic material used for gene doping, known as a transgene, scientists employ a commonly used amplification reaction that produces millions of copies of target segments between specific anchors in the transgene.To avoid contamination with a positive control, Baoutina’s team designed a reference material with enough similarity to the transgene to be amplified in the same reaction, but with a key difference that makes them distinct. The anchors in the reference material are the same as in the transgene, but are positioned differently in the sequence, resulting in amplified target segments of different sizes. This difference enables the reference sequence to be distinguished from the transgene, so it can serve as a positive control without the risk of cross-contamination with the test sample.  

Baoutina set out to test her team’s new method while they were designing a reference for the EPO gene. On analysis of one of their samples, they were able to detect one instance of cross-contamination that would have previously gone unnoticed. One of the blank samples used, containing all the reagents but no DNA, returned a positive signal.The team showed that it had become contaminated by DNA from the control EPO sample with which their reference was compared. 

In addition to demonstrating the strategy’s effectiveness, this highlights the difficulty of avoiding contamination, even in a national reference lab following the best practices. 

“This study does a very good job of highlighting the issue of cross-contamination and the need for methods to detect it,” says Perikles Simon, a gene doping researcher and professor at the Johannes Gutenberg University of Mainz who was not involved in the study. However, Simon points out that it won’t detect contamination from other sources, such as genetic material in the lab, and that techniques using this approach may not be sensitive enough to detect gene doping. 

“Gene doping itself isn’t a big issue because there isn’t a gene transfer technology at hand that could give a significant performance improvement,” he says. “Nevertheless, research related to gene doping has led to developments that could be helpful in other areas, such as detecting minimal residual disease,” or any few cancer cells that may remain in a patient following treatment and are difficult to detect.

References

  1. Baoutina, A., Bhat, S., Zheng., M., Partis, L., Dobeson, M., et al. Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy. Gene Therapy23, 708-717 (2016). | article

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