Projets de recherche

Code: T07D01RB 

Many studies are being carried out to develop methods for identifying exposure to growth factors such as human growth hormone, IGF1 and other related factors with potential application in sport doping. A number of these studies are based on the hypothesis that identification of genes and their protein products abnormally expressed in tissues treated with these factors can provide definitive evidence – i.e., signatures - for exposure to such growth factors. A number of related parallel studies are being conducted under the sponsorship of WADA and an enormous amount of information is being accumulated. However, to date no concerted effort has been made to collate and compare these large data sets and results to identify factors common to these studies that would represent the markers most likely to be informative in a drug testing setting. We propose to carry out a pilot informatics study to compare results from several completed and on-going WADA-sponsored studies.

Main Findings: 

Most of the current methods to detect doping rely on the use of chemical tests to identify drugs in the body. These methods are very powerful and often highly effective. Recent advances have enabled scientists to develop entirely new and potentially even more effective kinds of tests that are based on the detection of the broad changes that drugs cause in the genes and the proteins of affected tissues. These new methods can examine the ways in which all of the 25,000 human genes are affected by exposure to drugs and how those changes in genes affect the content of the even larger number of proteins in human cells. WADA has mounted a major research effort to use these approaches to develop detection methods for doping and is supporting work in more than a dozen laboratories scattered around the world aimed at finding genetic and protein changes in blood, urine and other body fluids and tissues that can definitively identify exposure to given drugs. Very promising results are coming from this effort and potentially useful "signature" gene and protein changes are being identified. A feature of these approaches is that they produce enormous amounts of information that require unified methods of analysis that are generally beyond the computing and informatics capabilities of individual laboratories. To ensure that the data are being evaluated in a consistent and effective manner, WADA has established a centralized bioinformatics laboratory that uses the most modern computational and analytical methods to harmonize the results of all the separate research laboratories to ensure that tests coming from this approach are highly sensitive, accurate and specific. The facility has succeeded in establishing an effective infrastructure and operating procedures and is beginning to obtain and analyze data from the research laboratories. WADA is confident that this more unified and centralized approach to doping detection will lead to far more effective tests that are rigorous, accurate and sensitive and that provide an effective and fair approach to protect athletes and Sport from doping.

Code: 06B06MG

This is a highly interdisciplinary and coordinated project that is aimed at understanding the molecular modifications induced by prolonged IGF-1 gene expression in the skeletal muscle after viral mediated gene transfer in rodent animals. The project will exploit the availability of AAV vectors expressing different IGF-1 isoforms to transduce skeletal muscles, a unique system that permites the long-term evaluation of the effects induced by the growth factor in vivo. In the treated animals, the signatures of these modifications will be analyzed by detecting the presence of the delivered transgenes in serum by quantitative real-time PCR and by analyzing the modifications of the proteomic pattern in muscle by advanced proteomics and mass spectrometry. These studies will be complemented by a parallel evaluation of the proteomic modifications induced by IGF-1 gene transfer in skeletal muscle satellite cells in vitro.

The project will be divided into three tasks.

Task 1. The overall purpose of this task will be the identification of a proteomic signature of IGF-1 gene transfer in muscle cells. The project will involve the development of animal models for AAV-mediated IGF-1 gene transfer. In particular, viral vectors expressing different IGF-1 isoforms will be produced and used to inoculate the tibialis anterior and femoral quadriceps muscles of mice and rats. At different times after transduction, proteomic analysis of the transduced muscles will be performed. These studies will take advantage of state-of-the-art technology 2-DE DIGE and will have the ultimate purpose of identifying protein patterns specific to the IGF-1 expressing muscles. These studies will be paralleled by the analysis of proteomic changes in human skeletal myoblasts after ex vivo gene transfer of the IGF-1 cDNAs.

Task 2. This task is aimed at the identification of novel peptides markers that might be exploited for anti-doping purposes. In particular, the project is aimed at the indentifications, quantification and qualitative assessment of the proteins differential expressed in muscle transduced with IGF-1. Differentially expressed or modified proteins will be analyzed using advanced mass spectrometry instrumentation, including MALDI-TOF/TOF, ESI-linear quarupole ion trap and high resolution FT-MS instrumentation.

Task 3. This task is aimed at the assessment of gene doping by monitoring the presence of exogenous gene fragments in animal serum. This possibility is based on different experimental and clinical reports that indicate that muscle exercise determines signficant damage to muscle cells, with the release of intracellular content into the bloodstream. At different times after in vivo IGF-1 gene transfer, both in resting conditions and during muscle exercise, serum will be analyzed for the presence of promoter-, vectore backbone- or cDNA-specific gene fragments by TaqMan-based Real Time quantitative PCR.

Main findings

The main findings are not available due to the sensitivity of the information and results developed in this project.

Code: 06B3MT

Growth hormone (GH) therapy has become a frequently employed strategy to fight dwarfism, and with the advent of biotechnologically produced recombinant GH, purity and sufficient amounts have been guaranteed. However, the need to prepare recombinant peptide hormones and repetitively inject drugs has led to gene therapy approaches that aim to generate GH-producing cells. Early attempts were based on myoblasts and fibroblasts modified to produce GH as growth hormone gene therapy does not necessarily need to be pituitary-specific because targets of GH are peripheral organs. The abuse of GH in sports has been a serious issue since years, and first assays enabling the discrimination between natural isoforms and recombinant GH have been established using a so-called differential immunoassay approach. More detailed information on GH isoforms used for the determination of GH administration is obtained using proteomics approaches employing 2D gel electrophoresis followed by mass spectrometric identification and characterization of GH and its fragments. In order to provide fundamental information for new methods complementing the commonly accepted assay and to provide basics for another option to cope with the problem of GH misuse, which includes administration of recombinant GH as well as gene therapy, GH isoforms shall be isolated of from human plasma followed by 2D gel electrophoresis, visualization, and subsequent mass spectrometric identification. Using this strategy, GH isoforms can be considered individually in terms of a profile, and more information on the variability and stability of single items is obtained. Owing to the well known fact that endogenously produced GH is down-regulated upon GH administration, the considerable change in GH isoform profiles should be detected using proteomics technology. Moreover, the fact that future GH gene therapy may include cells different from the pituitary, significantly different isoform profiles are possible and should principally be detectable employing the technology established in this project.

Main Findings

Growth hormone is widely abused by athletes for its anabolic and lipolytic as well as growth promoting effects. The presented method is capable of discriminating endogenous and recombinant growth hormone in plasma or serum samples if the concentration is high enough to detect endogenous isoforms which is the case for samples with a normalized spot volume of the main 22 kDa spot of >0.52. The capability to detect and visualize discrete isforms of hGH represents an important advantage for confirmatory HG analyses in sports drug testing and could compliment currently employed assays to reveal GH misuse. While endogenous samples show two or four isoforms on the blots, samples containing recombinant growth hormone lead to detection of one spot only. In addition to the volume limit, the order of appearance is considered and the detection of a more acidic 22 kDa spot, which is phosphorylated in endogenous samples but may be an artefact from protein production in samples containing recombinant growth hormone. The suggested discrimination limit of 0.52 may be corrected to even lower values if larger samples populations are analyzed. Additionally all growth hormone variants that are detected in the doping control method were identified by mass spectrometry approaches. This makes the doping control method even more powerful and allows a better evaluation of results.

Code: 06B17HM

Doping analysis is always in the misery of ethics, minimal invasive tissue collection and the establishment of sensitive, valid and reliable techniques to detect illegal use of forbidden substances in sports. Until now GC MS is the technique of choice to quantify the banned substances or their metabolites. But in the last years, modern techniques of molecular biology are in development to become alternative or supplemental choice to elucidate the abuse of diverse doping substances. Therefore, the combination of diversified screening of gene expression by high through put gene array analysis, evaluation of array data by quantitative RT PCR, information transfer to customized cheap gene array and the verification by established methods such as GC MS would be a useful synergy in the fight against doping. The following research application includes several subprojects. In appreciation of ethic recommendations of tissue collection, buccal epithelium, lymphocytes and leukocytes could serve as minimal invasive RNA source for the proposed comparative molecular biology analysis. The main focus is located on the evaluation of a fast and reproducible method to isolate sufficient amounts of intact total RNA out of the tissues by RNA-stabilizing chemical extraction techniques. The second step is to screen the two tissues for possible side effects by physical activity. Therefore, baseline results should be generated by a group of control sportmen. Following, three additional populations, asthmatics, anti aging patients (and/or patients with testicular hypofunction) and bodybuilders, should be tested for either the use of inhalative beta-2-agonists or anabolic steroids by gene array, RT PCR and GC MS in comparison to the control population. The main objective of the present study is to evaluate an innovative valid long term screening technique to elucidate candidate genes which are modulated by illegal substance and not by factors such as physical activity or circadian rhythm.

Main Findings

The current test results demonstrate that gene array analytics has not yet reached the aspired quality level to guarantee a constant reproducibility over time–particularly when involving batch removal. In addition, the low level of reproducibility may lead to the discrepancy between gene array and RT-PCR results. Realtime RT-PCR revealed high reproducibility. Results were subject to intense biological variation in relation to exercise load, time and hormone substitution. Additionally, hormone analyses from the saliva showed a potential method for direct evidence.

Future plans include: Expanded cluster analyses or new mathematical models possibly elicit further target genes that have to be validated via RT-PCR.

Code: 06A03AM

A method is proposed which will enable specific detection of adminstered recombinant erythropoietin (EPO) and ARANESP™ (Darbepoetin alfa) in urine, or other biological fluid such as blood, without regard for levels of EPO activity. This method is specific for recombinant erythropoietin and will not be subject to interference by endogenous erythropoietin. This method is based on the immunochemical detection of recombinant erythropoietin and ARANESP™ by specific detection of carbohydrate moieties which are unique to recombinant erythropoietin and ARANESP™ and are absent from the endogenous glycoprotein. This method is feasible due to the fact that the glycosylation of recombinant glycoproteins is dependent on the cell line in which the protein is expressed and the culture conditions. For these reasons, although possible, it is not necessary to determine levels of EPO activity to detect administration of rHuEPO to the athlete. This method will be rapid, cheap, and more specific than current methods in use. Glycozyme, Inc. in discussions with two different companies to produce the ELISA kit which could also be used to monitor the dosage of legitimate patients.

Main Findings

The immediate goal of this work was to obtain one or more monoclonal antibodies against the carbohydrate moiety of rHuEPO. The clones from the fusion screen are now monoclonal. The initial screen was against EPO and DG-EPO in which they did not bind DG-EPO as well as EPO by an amount that roughly correlates with the sialic acid content. The supernatants from five clones can be almost completely inhibited(at the concentrations tested) from binding EPO by Maackia amurensis lectin which specifically binds Nacetlyneuraminic acid α(2-3) galactose which is present on rHuEPO produced in CHO cells. (CHO cells do not have the enzymatic machinery to produce Nacetylneuraminic acid α(2-6) galactose.) The EPO molecule has been shown to have the carbohydrate chains on one side of the conformation with the antennary chains protruding so it is unlikely that the lectin binding the terminal sugars interfere with the protein chain. Finally, the supernatants from the twelve clones tested bind the glycopeptides GP-8 and GP-9 which were linked to KLH. This is a very important observation for several reasons. Our original rabbit anti-EPO antibody was obtained from a rabbit immunized with rHuEPO obtained from Amgen. This polyclonal antibody was shown to bind N-acetylneuraminyl α(23)galactose and galactosyl ß(1-4) N-acetylglucosamine (lactosamine) (Pazur, et. al., 2000). The rabbit antibody was shown to bind GP-9. The glycopeptides GP-8, GP-9 and GP-10 were obtained by treatment of the rHuEPO with Glu-c. Further, the rHuEPO from which the glycopeptides were isolated was obtained from another company which, according to some, would be considered a biosimilar rHuEPO even though it is still produced in CHO cells. So there then is indirect evidence that the monoclonal antibodies may bind one of the two antigenic determinants of the rabbit antibody. These results are very encouraging but these are still screening tests so now the antibodies must be produce in sufficient quantities to further characterize the carbohydrate binding specificity.

Code: 06A09FL

At the present time, anti-doping control for recombinant human erythropoietin (rHuEPO) relies on isoelectric focusing of ultra-filtered urine and double blotting of this hormone. This method results in an image of the isoelectric pattern of erythropoietin (EPO) present in urine and allows to differentiate between natural and recombinant hormone. Indeed, some of the isoforms composing the isoelectric profiles of natural urinary EPO are collocated with the isoforms of rHuEPO but others, more intense, present more acidic pI. This difference enables the detection of rHuEPO in urine but till now has no structural explanation. It is important to explore this structural aspect both from a fundamental scientific and anti-doping analytical points of views. We have undertaken investigations on this topic by studying the role of sialic acid residues in the isoelectric profiles of recombinant and natural urinary EPO. Our preliminary results show that in addition to sialic acid. Some other unidentified residues are responsible for the more acidic properties of natural hormone since after desialylation, the two hormones present different isolelectric patterns. This observation is very important since apparently, some isoforms corresponding to molecules devoid (or with very few) of these additional structures are observed in the asialopattern of rHuEPO but not in that of natural urinary hormone. The presence of these isoforms in a asialo pattern would thus constitute an absolute demonstration of the presence of recombinant hormone. It is thus envisaged to develop a confirmation test based on analysis of the asialopattern EPO. However, an essential condition to these investigations is obtaining specific antibodies well-recognizing the asialo-erythropoietin molecules and we have planed to produce such antibodies.

Main Findings

The objectives of this first part of the project was to obtain antibodies to asialo EPO in order to investigate the asialo EPO patterns obtained in various situations (negative, positive, unstable, atypical EPO profiles from urine samples) and from various recombinant drugs (Epoetin alfa, beta, omega, delta, Darbepoetin alfa). These antibodies would be used as primary antibodies in immunoblot following isoelectric focusing (IEF) of desialylated EPO. Immunization of two rabbits produced antiserum with very low titres that did not give any results on immunoblots. Immunization of 3 mice gave rise to better results and the most reactive mouse, after an additional injection of immunogen, was chosen for the fusion step. Sera and hybridoma (after purification of immunoglobulins by protein G affinity chromatography) were tested on immunoblot experiments. Whereas good results were obtained by dot blot, no results were obtained after IEF of desialylated Eprex. It is probable that the tested antibodies have conformational epitopes that are lost during IEF with urea. Since urea is necessary to perform IEF of EPO, the project was suspended.

Code: R06C01MS

Natural variability of endogenous substances in the body complicates the evaluation of the evidence with indirect markers of doping. Currently, reference ranges of markers of doping are derived from the analysis of data obtained from a large number of athletes. Unfortunately, such population based thresholds induce a lack of sensitivity for many markers, because they do not take into account the large heterogeneity that exists among professional athletes. For example, when the hematocrit is used as an indirect marker of blood doping, a threshold at 50% lets a margin large enough to athletes with naturally low levels (ex: 39%) to abuse from rhEPO or blood transfusions. In the same way, a limit at 4.0 or 6.0 for the ratio of testosterone over epitestosterone lets a margin large enough to the majority of male athletes to monitor their steroid profiles below this limit via the intake of exogenous testosterone. It has been shown recently that the combination of subject-based information, such as ethnical origin, gender, age or previous individual readings of the marker, with traditional population-based data can significantly enhance the sensitivity of most markers of doping (T/E, haemoglobin, ABPS,…). The approach used to integrate these different sources of information is based on Bayesian inference methods, and shares strong similarities with state-of-the-art approaches for the early detection of cancer. The aim of this project is to make this approach available to the anti-doping authorities for better decision making with any marker of doping.

Main Findings

The application called “Athlete Biological Passport Software” (ABP software) has been created. The ABP software is made freely available to any antidoping organization. The software has been distributed to some anti-doping organizations (sports federations, national anti-doping agencies, WADA accredited laboratories) and to the experts in charge of the evaluation of the haematological profiles during the ABP pilot project. The ABP software is divided into five main sections, with each section accessible through a dedicated data sheet: Athlete, Haematology, Endocrinology, Models, Results.

Publications

1. A forensic approach to the interpretation of blood doping markers. Sottas PE, Robinson N, Niggli O, Saugy M. Law, Probability and Risk 7:191-210 2008.

2. Les marqueurs indirects du dopage sanguin. Sottas PE, Robinson N, Saugy M. Revue Francophone des Laboratoires 401 : 27-38 2008.

3. The Athlete’s biological passport and indirect markers of blood doping. Sottas PE, Robinson N, Saugy M., In press in the Handbook of Experimental Pharmacology

Code: 06B14TK

Numerous genetic and/or pharmacological strategies exist that can increase muscle mass and strength. Many were developed to treat muscle diseases such as Duchenne’s muscular dystrophy (DMD). Importantly, these strategies have great potential to be abused by elite athletes seeking to gain a (unfair) competitive advantage, since they are, a) currently available and b) not detectable by current anti-doping protocols.

Prime amongst these are strategies for modulating the growth/development factor myostatin (GDF8); a negative regulator of muscle mass. We described an antibody-based blockade strategy to increase muscle strength and reduce muscle damage in the mouse model of DMD (Bogdanovch et al. 2002 Nature). Other blocking strategies have been described since our initial report, including a propeptide-based strategy (Bogdanovch et al. 2005 FASEB J). Of immediate concern regarding doping is the development of the myo-29 “humanized” myostatin-blocking antibodies by Wyeth, that are currently in human Phase II trials.

Currently, tests do not exist to detect myostatin blockade based ‘doping’ with antibodies, propeptides, or other reagents, underscores the need for rapidly developing a test to detect ‘doping’ not just the currently described reagents but also those that would be generated in the near future, including the use of gene doping approaches RNAi.

Thus, the challenge is to develop a standardized detection test that would be specific, sensitive and standardized enough to hold up to legal challenges that anti-doping agencies would almost certainly face by athletes caught using these tests. These challenges are not insurmountable; we propose to develop a robust and standardized assay for ‘total myostatin activity’ to serve this need.

Main Findings

The overall aims of this project are to develop tests to detect myostatin based 'doping' (blockade) and precluding abuse by elite athletes seeking to gain a (unfair) competitive advantage using myostatin-based doping. We have made excellent progress toward completion of this project and have cloned and developed the WADA-myostatin CAGA assay to serve as doping detection tests for this purpose. This luciferase-based assay emits light proportional to total myostatin activity and is used widely in research and industrial labs. We have developed two assays one in the C2C12 muscle cell line and one in the easier to grow HEK cells, hence it would be easy and efficient to transfer to WADA testing laboratories worldwide. It is important to point out that in myostatin-blockade strategies relevant for doping myostatin levels themselves are not altered rather the ability to activate the receptor or ‘activity’ is altered. Hence for detection of myostatin blockade in athletes the activity-based assay we developed i.e. the “WADA myostatin CAGA assay” will detect doping, however, assays based on detecting the molecule itself will not be useful, irrespective of their sensitivity. Technical analyses of the WADA-myostatin CAGA assay have been completed in our laboratory and the test is robust and valid based on the Z’ scores we obtained. We have also treated wild type mice and mdx mice with different myostatin inhibitors to obtain serum for validation of the myostatin-doping test if needed. A set of standards has been generated that are suitable for distribution to WADA laboratories worldwide. Given that our results demonstrate that the WADA-myostatin CAGA assay is sensitive, reproducible and robust we believe that the next logical steps would entail single-and double-blind testing of the assay with serum samples from athletes to develop normograms and developing standard operating procedures (SOP’s) for incorporation into current WADA testing protocols.

Code: R06D36WS 

Since midyear 2005 we detect with new mass spectrometric techniques in doping control samples a new unknown metandienone metabolite. First studies indicate that this metabolite can be detected for a longer time period than all other metandienone metabolites. The aim of the study is 1: to identify the structure of this unknown metandienone metabolite, 2: to investigate the excretion kinetics in comparison to other metandienone metabolites, 3: to synthesize reference substances and 4: to implement this substance in existing screening procedures for the detection of doping substances. The analysis of long-term metabolites can improve the fight against doping in the out of competition period in general, because misuse of anabolic steroids can be detected for a longer time.

Main Findings: 

Anabolic-androgenic steroids have been one of the most frequently detected drugs in amateur and professional sport. Doping control laboratories have developed numerous assays enabling the determination of administered drugs and/or their metabolic products that allow retrospectives with respect to pharmacokinetics and excretion profiles of steroids and their metabolites. A new metabolite generated from metandienone has been identified as 17a-methyl- 17b-hydroxymethyl-androst-1,4,13-trien-3-one in excretion study urine samples providing a valuable tool for the long-term detection of metandienone abuse by athletes in sports drug testing. The metabolite was characterized using gas chromatography - (tandem) mass spectrometry, liquid chromatography – tandem mass spectrometry and liquid chromatography – high resolution/high accuracy (tandem) mass spectrometry by characteristic fragmentation patterns representing the intact 3-keto-1,4-diene structure in combination with typical fragment ions substantiating the proposed C/D-ring structure of the steroid metabolite. In addition, structure confirmation was obtained by the analysis of excretion study urine specimens obtained after administration of 17-CD3-labeled Metandienone providing the deuterated analogue to the newly identified metabolite. 17a-Methyl-17bhydroxymethyl-androst-1,4,13-trien-3-one was determined in Metandienone administration study urine specimens up to 19 days after application of a single dose of 5 mg, hence providing a detection period extended by more than one week compared to commonly employed strategies.

Code: T06C05JS 

Non invasing imaging of gene expression will have a great potential usefulness in medecine (identify anomalous expression of gemes related to diseases, verify the result of gene therapy applications, etc) as an alternative to invasive biopsy approaches. Positron emission tomography (PET) and single photon emission computerized tomography (SPECT) technologies appear as the most sensitive imaging tools. A very important field of application of imaging tools. A very important field of application of imaing gene expression will be the prevention of the prohibited misuse of gene therapy in athletes. Some of the more studied approaches for imaging gene expression have been the the imaging of reported genes, introduced purposely through a gene transfer process, or the detection od the expressed protein when ir is an enzyme or a receptor. Neither of these characteristics take place in doped athletes, where the hormone (EPO, IGF-I, GH) is not an enzyme/receptor and where the practice is purposely attempted to be masked. An alternative of wider applicability for imaging gene expression is the detection of the actual outcome of transfection, that is, the mRNA being formed in unusual tissues after the gene transfer process. This approach is independent of the construct and the vector used for gene transfer and is applicable to any gene transfected to tissues not usually expressing the protein, such is muscle for EPO. The imaging of mRNA may be carried out by hybridization in the tissues of the mRNA molecules with suitable antisens oligonucleotides probes labeled for PET or SPECT detection. Some of the common problems when designing oligonucleotides for this purpose is the difficulty the may have in entering inro the cell in vivoand the stability of the mRNA because of the potential activation of RNAse H by the probe. In rhis regards, the recently developed Peptide Nucleic Acids (PNAs)  appear as one of the best alternatives to other obligonucleotides such as phosporothioates, 2'-O-methyl RNAs or 2'-fluoro-arabino nucleic acids. PNAs are expected to have high stability, strong hybridizinf properties, appropriate pharmacokinetic characteristics and the possibility to incorporate cell-penetrating peptides to allow cell entrance and accommodate positron or single photon emitting atoms (EG. 18f 11C, 123I) by relatively simple chemical treatments. In spite of all these advantages , the field of imaging gene expression by hybridization of mRNA by labeled obligonucleotides and analogs is not yet completely developed, and it is considered that a pilot project in animals (mice) is required before a more definitive effort be carried out for doping prevention. Accordingly, the pilot project proposed, addressed to image the presence of transfected EPO genes into muscle of mise, will follow the following steps: a) selection of target EPO-mRNA sequences suitable for hybridization b) synthesis of the anisense PNAs incorporating cell-penetrating peptides c) in vitro verification of the stability and hibridizinf properties of synthesized PNAs. d) labeling pf PNAs with PET ans SPECT emission atoms. E) transfer of EPO genes into mice muscle by electroporation. f) verification of successful EPO gene transfer and expression in trasfected mice g) imaging og gene expression in vivo by PET and/or SPECT after labeled-PNAs administration. h) conclusions and recommendations for futher steps.

Main Findings: 

The project IMAGENE (Non invasive molecular imaging of gene expression useful for doping control: Pilot study in animals after erythropoietin gene transfer) was approved by WADA on October 2004 for an initial period of one year. The results of this first period were presented to the WADA Committe on Gene Doping in Stockolm on December 2005. As a consequence of the recommendations of the meeting, an extension of the project was approved on March 2006. In this final report, an update of the overall project is presented. The hypothesis underlying the project was based on several assumptions. The first was that most of gene transfer processes produce the expression of an mRNA for the target hormone-protein in unusual cells or tissues. The second was that these mRNA molecules will hybridize with suitable antisense modified oligonucleotides such as PNAs introduced in the tissues expressing the ectopic hormone-protein. The third was that if a radiolabel of appropriate energy is associated to the modified oligonucleotides, the detection of the unusual hybridization may be carried out non-invasively from the outside of the body by suitable imaging technologies. Thus, the global objective of the project was to develop a pilot study in animals (mice) to verify the hypothesis for further potential extrapolation of the approach to humans in the future. The specific objectives were as follows a) to develop modified oligonucleotides (peptide nucleic acids (PNAs) with cell penetration properties (Tat-PNAs) to hybridize mRNA expressing transfected EPO. b) to label the Tat-PNAs with radiochemicals suitable for radiological external detection. c) to evaluate in vivo the imaging capability (PET/SPECT) in animals expressing EPO in muscle after a gene transfer process d) to develop recommendations for the development of similar procedures for the detection of EPO and other gene transferred doping hormone-proteins in humans. All the objectives of the IMAGENE project (out of PET studies) were accomplished