No abstract

As clinical gene therapy has progressed toward realizing its potential, concern over misuse of the technology to enhance performance in athletes is growing. Although 'gene doping' is banned by the World Anti-Doping Agency, its detection remains a major challenge. In this study, we developed a methodology for direct detection of the transferred genetic material and evaluated its feasibility for gene doping detection in blood samples from athletes. Using erythropoietin (EPO) as a model gene and a simple in vitro system, we developed real-time PCR assays that target sequences within the transgene complementary DNA corresponding to exon/exon junctions. As these junctions are absent in the endogenous gene due to their interruption by introns, the approach allows detection of trace amounts of a transgene in a large background of the endogenous gene. Two developed assays and one commercial gene expression assay for EPO were validated. On the basis of ability of these assays to selectively amplify transgenic DNA and analysis of literature on testing of gene transfer in preclinical and clinical gene therapy, it is concluded that the developed approach would potentially be suitable to detect gene doping through gene transfer by analysis of small volumes of blood using regular out-of-competition testing.

Vector biodistribution and clearance studies are essential in the development of gene transfer medicine. To provide reliable and accurate data, protocols for vector analysis must be optimized and validated. We addressed several parameters affecting the detection of gene therapy vectors in blood. Using an in vitro system based on plasmid DNA incorporating, as a transgene, complementary DNA for human erythropoietin gene, we developed and validated a suite of real-time PCR assays for the transgene splicing sites. The most sensitive assays detected the transgene present at 0.011% of the copy number of the endogenous erythropoietin gene in human genomic DNA at 100% specificity. Plasmid linearization incorporated with PCR resulted in an increase in assay sensitivity up to 4.5-fold without compromising analysis workflow. This allowed detection of five copies of transgene in a background of 0.4 μg of genomic DNA (or 0.0035% detectable transgene copies relevant to copies of the endogenous gene). Finally, desktop assessment of 18 DNA extraction protocols was undertaken and 5 kits were evaluated experimentally for extraction of nonviral vectors from blood. Three kits reliably detected 80 copies of the transgene in a milliliter of blood. Adoption of the described protocols will enable more reliable vector analysis in gene therapy and will assist in accurate interlaboratory comparison. The methodology will also facilitate detection of gene doping in sport, a potential new form of misuse of gene transfer technology.

Hypermethylation at promoter regions of tumour suppressor genes is diagnostic for many cancers. Many genomic regions that may be the targets for clinical diagnostic assays have been identified through use of measuring systems reliant on bisulphite conversion, but few of these promising markers are in clinical use. The comparability of a widely used DNA methylation measuring system involving bisulphite conversion was evaluated by supplying three experienced centres with methylated DNA reference material mixtures that were independently prepared and characterised by mass spectrometry and high-pressure liquid chromatography. A replication scheme was designed to evaluate reproducibility of key analytical steps within and between laboratories by regression analysis. In general, methylation was underestimated and methylation ratio values were highly variable. The difference in methylation ratio between CpG sites was the key contributor to variable results. The CpG site effect followed a similar pattern at all centres and at all methylation levels examined indicating that sequence context had a major effect on methylation ratio measurement using the bisulphite conversion process. The magnitude of underestimation combined with the variability of measurements between CpG sites compromises the concept of measuring genomic regional methylation by averaging the methylation ratios of many CpG sites. There were no significant differences in replicate bisulphite conversions or sample work-up and instrument analysis at each centre thus making this technique suitable for comparative intralaboratory investigations. However, it may not be suitable for a routine diagnostic assay without extensive standardisation efforts.