Development of Reliable Blood Tests for the Detection of Gene Doping after Intramuscular Injection of Recombinant Adeno-Associated Viral Vectors

Code: 11C9RS

Aim 1: Assay optimization

We will evaluate a combined assay format that is based on real-time PCR and ITC readout with a nested PCR pre-step to improve sensitivity. The current single-step qPCR assay has a sensitivity of 3 copies for the macaque Epo cDNA in the presence of 500ng - 1 OOOng macaque gDNA. A qPCR assay has been developed to the homologous locus in the human Epo cDNA with a sensitivity of 10 copies in 500ng human gDNA. A nested PCR assay has been developed for the human Epo cDNA with a sensitivity of 10 copies in 1250ng of human DNA. We will evaluate the enhanced sensitivity of nested PCR combined with the automation and rapid results of qPCR with the aim of reaching a sensitivity of less than 5 copies in 500ng genomic DNA. We will also examine the feasibility of utilizing the ITC assay format with the nested+qPCR approach. Once the assay is developed and tested using plasmids and virions spiked into gDNA and human blood, we will evaluate samples obtained directly from transduced macaques, and macaque PBLs spiked into na"fve human blood. The SV40 polyA site, which is not present in the human genome, but will be present in the rAAV vector used here, is an ideal target since there is no competition with endogenous sequences. The SV40 assay was previously developed [1Jfor the macaque studies and is considered to have the best sensitivity; it will serve as a reference for optimizing the hEPO Exon3-4 assay. Controls (ITC format) will be developed for each assay.

Aim 2: Assay validation

New or revised clinical diagnostic methods must be validated: defined as the process by which it is established, by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical application. Components of validation involve quality control (QC) and quality assurance (QA). We will perform analytical validation of the human Epo assay to ensure specificity, sensitivity, and ruggedness (multiple labs, multiple operators, repeats and replicates). This will involve drafting test records, along with assay specifications, reagent preparation logs, and a validation plan, and executing the assay(s) several times with several replicates in Florida and Mainz. Data will be captured and evaluated statistically to determine the assay performance criteria. For pre-clinical performance validation, we will evaluate spiked (using plasmid DNA) human blood samples with respect to: sex, age, race, medical history (if available), exercise status, general health, and prescription drugs (if known). The rate of false positives and false negatives will be established by testing a statistically significant number of samples. We will screen up to 100 blood samples (50 for each lab in Florida and Mainz) for false positives in order to get an idea of the specificity of our procedures. The Simon Lab will contribute 100 human samples (Germans), and if available, substitute some of the German samples with samples of other ethnicities.

The assay validation activities support the documentation for eventual regulatory submission and eventual manufacture of an in vitro diagnostic test (IVD) kit. Ultimately, data from the assay development, analytical validation, and pre-clinical performance validation studies will be documented along with a description of each kit (with all required components) in a form suitable for regulatory submission.

As for regulatory capability and expertise, in addition to his faculty appointment and research activities described in this proposal conducted in the Department of Molecular Genetics and Microbiology, Dr. Snyder is the Director of the Center of Excellence for Regenerative Health Biotechnology at University of Florida (UF CERHB, http://www.cerhb.ufl.edu/ ). Established in 2003, the UF CERHB is committed to translational research in many different biotechnological fields including viral vectors for gene therapy. UF CERHB's biopharmaceutical development operation, offers a wide range of cGMP-compliant biopharmaceutical manufacturing and testing services to the biotechnology industry and to biomedical research institutions. Furthermore, Dr. Moullier runs a similar operation in Nantes called ATLANTIC BIO GMP (ABG). Resources are available (on a fee-for-service basis) at UF CERHB and ABG, Nantes for anti-doping PCR assay kit manufacturing.

Main Findings

It is crucial to develop reliable test methods for the detection of gene doping, given that some members of the sports community are seeking for gene transfer technology to improve exercise performance. Based on previous work we established a ´nested real-time PCR´ assay. This is an assay based on the PCR technology which is used in criminology and well-known in the court of law for the identification of genetic material in samples from sites of crime. We adapted this technique to detect single erythropoietin (EPO) DNA molecules that had been introduced into the body by means of gene transfer. Due to its high sensitivity, our technique is principally able to detect genetic modifications of certain parts of the body in conventional blood samples. Here we aimed at providing evidence of the high sensitivity and specificity of our refined procedure that now enables quantification of the artificial DNA in the blood stream.

Through an inter-laboratory study between Nantes (France), Gainesville (Florida, USA), and Mainz (Germany) two nested real-time assays were validated and utilized for an in vitro blinded study. Both assays showed unique sensitivity and specificity in a large dynamic range. According to the nested assay protocol the detection of ~1 copy of circular transgene molecule in a background of 500 ng gDNA is possible reliably. In a blinded in-vitro study the reliability of the assays to detect between 1000 – 2 copies of transgene molecules and non-template control was proven.

To validate the suitability of the nested real-time assays for the detection of gene doping a non-human primate study was conducted. Two macaques were injected intramuscularly with a recombinant adeno-associated virus serotype 8 (rAAV8) vector harboring human EPO cDNA sequence. One macaque served as non-injected control. The vector was promoterless to avoid transgene expression. Following the injection of 2.5 x 1011 viral genomes/kg, the transgene molecules were detectable 8-14 weeks.

The high sensitivity of the nested qPCR assay along with the specificity for transgene detection is essential for gene doping surveillance. The assay can be adapted to other gene doping candidate genes.