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

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.

A polymerase chain reaction-based methodology to detect gene doping

“…This relates to a very recent report demonstrating that intramuscularly injected conventional plasmid is rapidly eliminated (Ni et al 2011). Equally we noted that the signal for exogenous FAK was importantly reduced in transfected soleus muscle 7 days after transfection.…”

“…While the detection of genetic modiWcation has been shown for virus-mediated transfection of skeletal muscle with expression construct for the secreted hematopoietic factor erythropoietin (Beiter et al 2011;Ni et al 2011) it is unlikely that this transfection technology would be a method of choice. This is explained by various drawbacks including the induction of immune responses that reduce the success of repeated applications, the costs of production of GMO quality vectors and possibly fatal consequences due to the virulence of the approach (Raper et al 2003).…”

Loop‐mediated isothermal amplification (LAMP) as an alternative to PCR: A rapid on‐site detection of gene doping

“…[29] Using the same approach, it was demonstrated that detecting doping transgene in blood of nonhuman primates was possible. [25] Indeed, intramuscular injection of a conventional plasmid and rAAV vectors resulted in the presence of DNA that could be detected at high levels in blood and that rAAV genomes can persist for several months in WBCs.…”

“…[25] However, due to its high invasiveness, muscle biopsy would be impossible for sample harvesting. Direct injection into a target tissue has been considered as an approach which leads to entry of minutes amounts of transgenic DNA into the blood stream.…”

“…[1] Various direct detection methods based on nested and real-time polymerase chain reaction (PCR) have been proposed for the detection of gene doping practices. [24][25][26][28][29][30] Those approaches are based on subtle differences between the sequences of a doping transgene and its endogenous counterpart in genomic DNA (gDNA). Indeed, the gene sequences of transgenic DNA (tDNA) do not contain intron sequence parts of the gDNA which allows the detection of trace amounts of a transgene in a large background of gDNA.…”