Gene doping in cycling: Banned, but used?

Danilo Di Luca, the winner of the 2007 Giro d'Italia, was given life ban after testing positive for EPO

Danilo Di Luca, the winner of the 2007 Giro d’Italia, was given life ban after testing positive for EPO

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“We were contacted by numerous athletes, even coaches. They didn’t understand that we were still at an early stage in terms of gene therapy moving to humans”

–          Prof Lee Sweeney,Wada expert advisor on gene doping & director for center for orphan disease research & therapy, University of Pennsylvania – BBC, January 2014

The doping authorities have been preparing for this battle for over 10 years [back in 2003 the World Anti-Doping Agency banned gene doping]. The idea is simple: you alter an athlete’s genetic makeup to invent a new competitor that is stronger or faster. I have spoken with a number of coaches on whether this is indeed going on today; whether synthetic genes are being introduced to a

Gene Doping has been a topic of a number of sports blog posts, including this from Potomac Post; click to see post

Gene Doping has been a topic of a number of sports blog posts, including this from Potomac Post; click to see post

cyclist’s genome to make them produce their own (and more) Erythropoietin/EPO (for example). Is this scientific know-how accessible to the peloton today? Would the doping agencies have the tools – both legally & biologically – to detect & prosecute it if it was?

On a side note, some bioethicists have argued if genetic enhancement is possible should it be banned? This argument was placed in ‘Nature’ in 2012 – Olympics: Genetically enhanced Olympics are coming – the authors predicting that future Olympic Games may allow handicaps and gene therapy for people born without genes linked to athleticism. This has even been discussed legally – Eligibility of athletes receiving necessary gene therapy: the Oscar Pistorius case as procedural precedent.

There is a growing amount of evidence/examples that show genetic enhancement is already a part of professional sports. For instance, almost every male Olympic sprinter and power athlete ever tested carries the  allele, a variant of the gene ACTN3. Eero Mäntyranta, a seven time cross-country skier Olympic medalist, had a mutation in the gene EPOR that caused him to produce extra red blood cells, boosting his oxygen-carrying capacity by 25–50%. These naturally occurring mutations bring to question the current fairness of the sporting industry.

I think this point would be an interesting subject for another day, but let’s first look at whether gene doping is even possible. To address this topic; I want to break it down into two sections:

1. What gene doping could be available for cyclists

With all the focus on the Armstrong Lie & other veteran cyclists indulging in old doping habits, the regulatory authorities must also investigate considerable resources in the possibility of future scandals involving a new generation of drugs. This section looks at the options available for cyclists; and when I say ‘options’ experimental therapies that have only been tested in animals – do note if it works in mice it won’t necessarily translate to humans, case in point of several cancer drugs – or in limited early clinical trials (few proven human application is demonstrated by section 2).

2. A comprehensive overview of what the known science is in the pursuit of gene therapies for medical treatment. Enough of the hype, this is the proven applications of gene therapy. And I have made a boring list to avoid any hype/confusion/lack of information. Do note that these clinical trials are with experimental therapies for often very seriously ill patients, who are willing to take the health risk. For many persons, a risk such as this for a young, ultra-healthy individual who’s competing at the peak of their career is incomprehensible, yet as described in CyclingTips’ article on ‘The new EPO? — GW1516, AICAR and their use in cycling’, athletes are so determined to win that WADA took the ‘rare step of warning “cheats” to ensure that there is complete awareness of the possible health risks.

[Video – Panel discussion at AAAS of “Cracking Your Genetic Code]

 

[Section 1] What Gene Doping is available for cyclists

Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein. Fascinatingly the therapeutic technique is moving from the conceptual stage to technology development to their application in patients for clinical trials for a variety of patient disorders.

How Gene Therapy Works

How Gene Therapy Works

An article last July (2013) in the British Journal of Sports Medicine – Gene doping: an overview and current implications for athletes – stated that there was no conclusive evidence that it was being practiced in sport, however ‘given that gene therapy techniques improve continuously [as we just saw above] the likelihood of abuse will increase’. From a literature search of relevant proteins in development between 2006-20011, the authors found that the list of potential candidates included:”erythropoietin, peroxisome proliferator-activated receptor-delta (PPARδ) & cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; insulin-like growth factor), growth hormone, myostatin, vascular endothelial growth factor, fibroblast growth factor, endorphin and enkephalin, α actinin 3; and that for efficiency reasons there would be a preference for inserting gene target combinations rather than single gene doping products.

Please note many of these gene techniques are in the preclinical stage and so their application in athletes is not only risky but also at the cutting edge of science.

Next Gen Blood Doping – Erythropoietin & ‘Hypoxia inducible factors’

As detailed by the comprehensive article on CyclingTips – The Future of Blood Doping – EPO Gen Doping – despite the ban on gene doping by the International Olympic Committee in 2003, athletes may be using gene-targeting treatments.

Repoxygen is a gene therapeutic at preclinical development, constructed as a viral gene myostatin_mousedelivery vector carrying the human EPO gene under the control of a ‘hypoxia control element’ (HRE). Therefore Repoxygen acts as a transcription factors (proteins which influence gene expression in the cell) an important mediators in the cellular response to hypoxia (where the body is deprived of adequate oxygen). The gene therapy is being developed by Oxford Biomedica to treat anemia and has induced syntheses of EPO in mice muscle tissue (normally EPO is produced in the kidneys & liver).

In 2004, German track coach Thomas Springstein was caught trying to get hold of Repoxygen and at the 2008 Beijing Olympics, an unidentified Chinese doctor was caught by a German journalist in offering stem cell injections to swimmers.

Increased Endurance – PPAR-β & PEPCK-C

The 2013 Gene doping paper identified PPAR-β & PEPCK-C as the two therapies that had the highest potential for abuse. PPAR-β (or delta) encodes for a variety of biological processes and is being investigated for its association with several chronic diseases – diabetes, obesity, atherosclerosis and cancer. As described in this review – Gene doping: Of mice & men – PPAR-delta is associated with the formation

'Super' mouse on treadmill - how endurance is tested

‘Super’ mouse on treadmill – how endurance is tested

of slow-twitch muscles and in mice has shown to lead to a 67% & 92% improvement in running time & distance, respectively. CyclingTips quite rightly identified this as – The New EPO? – GW1516, AICAR and their use in cycling – and the compound has been prohibited by WADA since 2009 due to its potential to artifically increase endurance. As such, sports drug testing laboratories have established detection methods to identify the intact substance and/or its metabolites.

The same ‘Of mice & men’ review, described PEPCK or insulin-like growth factor 1 (IGF-1) as a mediator peptide for human growth hormone and also has an independent role in muscle growth & shift to the glycolytic metabolism. In ‘Super mouse models’ this gene [with a viral vector] caused a 14% increase in strength in young mice & 27% in older mice – so for an athlete this could not only mean enhanced performance but a longer competitive time frame.

Increased Muscle Mass – Myostatin

Principles of ACE031 (not a gene therapy)

Principles of ACE031 (not a gene therapy)

Nearly a decade ago, Lee Sweeney – physiologist at the University of Pennsylvania – wrote an article for Scientific America on how gene therapy for restoring muscle lost to age is being investigated by elite athletes trying to enhance performance. It is also worth noting that manipulation of myostatin is not limited to gene therapy; Acceleron Pharma & Shire only last year confirmed they were holding a promising the injectable ACE-031 – a synthetic activin receptor type IIB, which picks up the myostatin. 

Belgian Blue bulls have huge amounts of muscle because of a mutation in the gene that codes for the protein myostatin - which normally limits muscle fibers

Belgian Blue bulls have huge amounts of muscle because of a mutation in the gene that codes for the protein myostatin – which normally limits muscle fibers

In 2000 American researchers published – Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation – in which they had genetically manipulated mice so that their cells started to produce dominant-negative myostatin [dnMS].Dominant-negative myostatin deactivates the normal muscle-growth inhibiting myostatin and this intervention made the mice more muscular.

This research has now produced several muscle-building drugs – including transcription activator-like effector nucleases (TALENs) to disrupt myostatin expression at the genome level – now being tested in people with medical problems, including muscular dystrophy, cancer and kidney disease. The drugs all work by blocking a substance called myostatin that the body normally produces to keep muscles from getting too big.

One recent human application has been given the go ahead the 2013 $300,000 Phase I SBIR grant from the NIH’s National Institute of Arthritis and Musculoskeletal and Skin Diseases, towards biotech start-up Milo Biotechnology clinical testing of AAV1-FS344; a gene therapy intended to up-regulate follistatin, a protein that blocks myostatin. The follistatin therapy is intended for the treatment of muscular dystrophy.

Furthermore Growth & Differentiation factor-11 (GDF11) – Myostatin is GDF8 – and activin receptor type IIB (ActRIIB) could also be targeted in the coming years; do note this 2013 paper Regulation of GDF-11 and myostatin activity by GASP-1 and GASP-2 & Targeting the Activin Type IIB Receptor to Improve Muscle Mass and Function in the mdx Mouse Model of Duchenne Muscular Dystrophy (2011).

Increase in Pain Endurance – Endorphin & Enkephalin

Lance armstrong painAs detailed in the excellent review – The use of genes for performance enhancement: doping or therapy? (2011) – genes encoding for endorphin and enkephalin are prominent contenders for performance enhancement in competitors (no pain, no gain; suffering in cycling etc). When these molecules are expressed they bind to opoid receptors promoting analgesic effects and therefore pain relief (enkephalin is a pentapeptide involved in the regulation of nociception – nervous system of noxious stimuli/potentially tissue damaging events & endorphins – as you may know from your workout high – functions to inhibit neurotransmitters). Promising studies have been published in animal models – Gene therapy for chronic pain management.

Increase oxygen supply – Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) promotes the branching of a pre-existing vessel, in a process called angiogenesis; which allow for more rapid & effective diffusion of oxygen into the tissues. Investigators are currently developing innovative VEGF gene therapies for ischemic

Doping targets, as summarized in the paper - The use of genes for performance enhancement: doping or therapy? - Click to read this great paper

Doping targets, as summarized in the paper – The use of genes for performance enhancement: doping or therapy? – Click to read this great paper

cardiovascular disorders, including acute myocardial infarction, chronic cardiac ischemia, peripheral artery disease and stroke. As mentioned in this 2012 paper – VEGF gene therapy: therapeutic angiogenesis in the clinic and beyond – these VEGF-based therapies have already reached clinical experimentation.

[Section 2] Known Clinical Science & Medical applications 

To date, over 1800 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. If you are interested in the most notable medical advancements of gene therapy please read on – this will give you a picture at what development stage the most advanced R&D departments are. This summary will give the casual journalist an overview on how close the techniques of gene therapy are to being applied in athletes.

You will note that the most advanced clinical studies are only in small groups of patients, with the majority having been tested in the last 12 months. Note this section is very science heavy!

1. Gene therapy for ADA-SCID: defining the factors for successful outcome – two clinical studies have shown the long-term success of hematopoietic autologous stem cell (HSC) gene therapy in correction of Adenosine deaminase (deficiency) -Severe Combined Immune Deficiency (ADA-SCID). The therapeutic gene called ADA was introduced into the bone marrow cells of patients in the laboratory in Italy, followed by transplantation of the genetically corrected cells back to the same patients. The immune system was reconstituted in all six treated patients without noticeable side effects, who now live normal lives with their families without the need for further treatment.

2. Chronic Granulomatus Disorder (CGD) – Haematopoietic stem cell transplantation (HSCT) appears to offer a cure. In a study last year which looked at the outcome of HSCT in 14 Swedish patients with CGD to that in 27 patients with CGD who were given conventional treatment; thirteen of the 14 transplanted patients are alive and well in the first treatment whilst seven of 13 who were treated conventionally died from complications of CGD at a mean age of 19 years, while the remaining patients suffered life-threatening infections.

3. Hemophilia – A landmark study published in 2011, Nathwani et al demonstrated successful conversion of severe hemophilia B to mild or moderate disease in 6 adult males who underwent an infusion of an adeno-associated viral (AAV) vector expressing factor IX. These 6 subjects have now exhibited expression of FIX at levels ranging from 1% to 6% of normal for periods of > 2 years. At the end of last year, researchers at the UNC School of Medicine and the Medical College of Wisconsin found that a new kind of gene therapy led to a dramatic decline in bleeding events in dogs with naturally occurring hemophilia A. Additionally the team figured out a potential way around the antibody response by using a plasmapheresis machine & a blood-enrichment technique, to isolate specific platelet precursor cells that have hemophilia A. The team then engineered those platelet precursor cells to incorporate a gene therapy vector that expresses factor VIII. The engineered platelet precursors back into the dogs and as the cells proliferated and produced new platelets, more and more were found to express factor VIII. It seemed that at least in dogs, nature took over and platelets were naturally discharge at sites of vascular injury.

4. A number of clinical trials in genetic disorders will soon be launched including congenital blindnesslysosomal storage disease and muscular dystrophy, among others]

5. Cancer – To date, only one immunotherapeutic that could be considered a vaccine, Dendreon’s Provenge, has gained U.S. marketing approval. But even with late-stage clinical trial failures accrue, multiple gene therapy strategies are still being developed to treat a wide variety of cancers, including suicide gene therapy, oncolytic virotherapy, anti-angiogenesis and therapeutic gene vaccines. These approaches are a direct result of unprecedented discoveries of genes and pathways involved in tumorigenesis revealing potential targets for gene and cellular therapies. With this knowledge in the literature, researchers are now studying novel approaches such as siRNA delivery to block a critical pro-growth pathway or delivery of a gene coding for a pro-apoptotic inducer can be realized. What gene therapy does allow is a tailored, targeted approach that has the potential to improve efficacy and minimize toxicity.

At the start of 2012, predictions had been that three immunotherapy products could reach the market in the 2014–2015 timeframe this included:

Biovest International’s BiovaxID, an autologous idiotype lymphoma cancer vaccine (Id-KLH/GM-CSF), projected to be approved in Canada and the EU in 2013–2014 and in the U.S. in 2017. In 2012, the FDA denied marketing approval for BioVaxID, requiring that the vaccine undergo another Phase III trial. However in January 2014, the company announced that the European Medicines Agency (EMA) accepted its marketing authorization application for BiovaxID, thus beginning the review process intended to secure approval for the treatment of non-Hodgkin’s follicular lymphoma in patients [who have achieved a first complete remission].

Vical‘s Allovectin-7 [plasmid-based immunotherapeutic that expresses the HLA-B7 and β2 microglobulin genes, forming a major histocompatibility class I complex] in melanoma had failed to meet key endpoints (statistically significant improvement at time intervals when compared with first-line chemotherapy) in a Phase III trial in patients with stage III/IV metastatic melanoma and lead to the company dropping the product.

GlaxoSmithKline’s MAGE A3 [incorporating recombinant MAGE-A3 protein and a novel immunostimulant, AS15 (a combination of QS-21 Stimulon adjuvant, monophosphoryl lipid A, and CpG7909, a TLR-9 agonist] failed its first co-primary endpoint (last September) in a Phase III study for melanoma, failing to produce better results than a placebo in enhancing disease-free survival. The company says it will carry on with the study to determine whether the second primary endpoint—testing the immunotherapy in a genetically defined subpopulation—will provide better efficacy, with data expected in 2015.

Looking back down the pipeline, the industry will be watching these trials closely:

Introgen’s Phase III trial of Ad.p53 for head and neck cancer. The adenovirally-mediated p53 gene therapy is aimed at encoding the p53 protein which is one of the most intricate elements in the apoptotc (cell death) signalling cascade – a mutation in which leads to a decreased ability of a cell to apoptose & results in tumour growth.

Aduro Biotech announced safety and efficacy data from its Phase II clinical trial of a two-vaccine approach, suggesting that 93 pancreatic cancer patients demonstrated a statistically significant survival benefit in patients receiving the combination of GVAX Pancreas and CRS-207 cancer vaccines compared to GVAX Pancreas vaccine alone.

Virtual drug developer Madison Vaccines Inc. (MVI) says it raised $8 million to further its efforts to advance its MVI-816 DNA vaccine for prostate cancer beyond basic research and has completed Phase I in early-stage patients. The trial demonstrated that MVI-816 was safe and that the product induced antigen-specific CD8+ cytotoxic T-cell immunological responses and prostatic acid phosphatase (PSA) doubling times in more than 30% of patients.

Agenus announced last September (2013) that an analysis from a Phase II trial in patients with newly diagnosed glioblastoma multiforme (GBM) treated with the company’s Prophage Series G-100 (HSPPC-96), a vaccine [autologous therapies derived from cells extracted from the patient’s tumor] used in combination with the current standard of care (radiation and temozolomide), showed an almost 18-month median progression-free survival, representing a 160% increase versus current standard of care alone.

Bavarian Nodric‘s showed promising clinical evidence from it’s phase 1 trial combining PROSTVAC plus ipilimumab (previously published in The Lancet Oncology) and announced a few days ago at the American Association for Cancer Research (AACR) Annual Meeting in San Diego

NewLink launched a first in human Phase 1 clinical trial of HyperAcute Renal immunotherapy in patients with metastatic renal cell cancer. HyperAcute Renal Immunotherapy is comprised of two allogeneic renal cell cancer cell lines engineered to express the murine alpha(1,3)GT gene.

 6. Neurodegenerative Diseases – Recent progress in gene therapy has allowed for novel treatments of neurodegenerative diseases such as Parkinson’s Disease and Huntington’s Disease, for which exciting treatment results have been obtained in appropriate animal models of the corresponding human diseases. In January a study in 15 Parkinson patients (all in advanced stages) at Imperial College London using a treatment called ProSavin, a modified virus to deliver three genes into the striatum [a part of the brain that controls movement], described a 30 per cent improvement in movement tests with no serious adverse effects. The gen therapy is designed to boost the production of dopamine, a chemical that becomes deficient in patients with Parkinson’s, and is seen as superior to current treatments that only boost the neuroendocrine transmitter temporarily.

7. AIDS – Gene therapy is being used to mimic a rare but natural mutation that makes about 1% of the population resistant to the most common strains of HIV. The mutation results in a lack of CCR5 which is a cell surface receptor used by HIV to get inside our immune cells. By editing cells harvested in patients and then infusing them back into the patient (10 billion at a time). A clinical trial involving 10 patients showed that 4 showed lasting long time effects and their antiretroviral therapy taken off. One patient’s HIV levels fell so low that they could no longer be detected and a follow up investigation showed that he had already inherited a rare resistance mutation from one of his parents, thus giving his immune system an advantage.

8. Other acquired diseases – The same gene therapeutic techniques are being studied in the treatment of Influenzacardiovascular disease and gene therapy , among others. Some of these have entered, or will soon be entering, into early phase clinical trials.

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