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Unit 5 pre-release

Unit 5 pre-release. Scientific article 2011. Text book reference 8.7 – genetic modification. Muscles, genes and gym in a bottle. Gene therapies to treat genetic disease can be abused by athletes Gene-doping to grow bigger muscles

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Unit 5 pre-release

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  1. Unit 5 pre-release Scientific article 2011

  2. Text book reference 8.7 – genetic modification Muscles, genes and gym in a bottle • Gene therapies to treat genetic disease can be abused by athletes • Gene-doping to grow bigger muscles • Erythropoietin to increase oxygen carrying-capacity of the blood P2 Muscles, genes and gym in a bottle

  3. Erythropoietin .. • Mantyranta (Finnish cross-country skier) 1964 won two gold medals • A genetic mutation in the gene producing receptors for erythropoietin caused his blood to have 25-50% more red blood cells than normal P2 Muscles, genes and gym in a bottle

  4. Text book reference 7.6 – Action of hormones How Erythropoietin functions • Erythropoietin (epo) is a glycoprotein hormone • It is produced and released by the kidney when oxygen level of the blood are low • Epo then travels in the blood to the bone marrow • It combines with the erythropoietin receptor (EpoR) on the cell surface membrane of red blood cell precursors causing them to increase the number of red blood cell produced P2 Muscles, genes and gym in a bottle

  5. Text book reference 7.6 – Action of hormones Action of erythropoietin Hormone receptor for erythropoietin Hormone - erythropoietin on red blood cell precursor in bone marrow Text book reference 7.6 transcription factors activates transcription factors leading to mRNA and translation to proteins which cause increase in red blood cell manufacture (also inhibits apoptosis) P2 Muscles, genes and gym in a bottle

  6. Text book reference 7.6 – negative feedback Action of erythropoietin • A feedback mechanism means that when blood oxygen levels return to normal… • …. the kidneys no longer produce epo • …. therefore the epo receptors are no longer stimulated • ….and no extra red blood cells are produced P2 Muscles, genes and gym in a bottle

  7. Text book reference 7.6 – Action of hormones What mutation did Mantyranta have? Hormone receptor for erythropoietin Hormone - erythropoietin • Mantyranta’s mutation meant his EpoR receptor was never turned off so he continually made new red blood cells • This is a very rare mutation on red blood cell precursor in bone marrow activates transcription factors leading to mRNA and translation to proteins which cause increase in red blood cell manufacture (also inhibits apoptosis) P2 Muscles, genes and gym in a bottle

  8. Text book reference 8.7 – genetic modification Epo doping Text book reference 7.6 Performance-enhancing substances • Injecting epo means anyone can increase their red blood cells • 1989 Biotechnology company Amgen began marketing a form of epo produced by recombinant bacteria for treating severe anaemia from suffered by AIDS and kidney patients • It then began to be exploited by athletes • Employee of Festina cycling team found with car load of performance-enhancing drugs including epo at 1998 Tour de France • Swiss rider Alex Zulle ‘Doping is part of the business of cycling’ P2 Muscles, genes and gym in a bottle

  9. Text book reference 7.6 Performance-enhancing substances Secret weapon Text book reference 7.2 oxygen required for ATP production in respiration • Widespread doping? • Australian Open tennis • Cross-country skiing • Football • Track and field athletics • Epo rumoured to make athletes run 20% faster • Charles Yesalis – epidemiologist Pennsylvania State University, ‘we only reward winners and drugs work’ • Problem could get worse if athletes could insert a gene to make their bodies produce epo Text book reference 7.1 ATP required for muscle contraction P3 Secret weapon

  10. Text book reference 2.7/8.7 genetic engineering with viruses ‘gene therapy’ for athletes • Epo needs injecting several times a week, ‘gene therapy’ would give them the equivalent of Mantyranta’s super-gene • This gene therapy is already under development by several academic groups and biotech companies for anaemia e.g. Avigen • Use viruses as vector P3 Secret weapon

  11. Text book reference 6.3 Body’s response to infection Adenovirus as vector • Genes making it pathogenic removed • Advantages as a vector • Large size so can carry big genes • Disadvantages • Easily recognised and destroyed by immune system • Will immune system destroy it before the gene is delivered? P3 Secret weapon

  12. Gene therapy with adenovirus • Avigen have patented adeno-associated viruses (AAVs) for delivering epo • Smaller than adenovirus • Carries a smaller load • BUT less vulnerable to attack from immune system P3 Secret weapon

  13. Gene therapy success • Both viruses have shown ‘exceptional results’ • 1997 (Leiden , University of Chicago) - adenovirus to deliver epo gene to mice and monkeys • Injected into muscles • Infiltrated cells • Inserted epo gene • Cells pumped out epo • Mouse hematocrits (proportion of blood volume made up of red blood cells) up from 49% - 81%, lasted over a year • Monkey hematocrits up 40% - 70%, lasted 12 weeks P3 Secret weapon

  14. Hematocrits • Proportion of blood volume made up of red blood cells • Typically • males.......... 40-50% • females....... 38-45% • athletes........ > 50% • Any activity or condition that consistently lowers oxygen levels in the blood will cause an increase in erythropoesis and a subsequent rise in the hematocrit. • Factors that will raise the hematocrit include: • Exercise. regular aerobic exercise raises the hematocrit. • Living at high altitude • Injection of recombinant erythropoetin P3 Secret weapon

  15. More on gene therapy • Biotech company Chiron reported similar results in 1998 using AAVs to deliver epo gene to two BABOONS (mistake in paper) • Hemaocrits 38/40% to 62/75% remained for 28 weeks of study • Risk free?? • No, 18 yr old patient receiving gene therapy for rare liver complaint died after adenovirus used to deliver gene • Currently unsure what went wrong so reviewing safety P3 Secret weapon

  16. Gene therapy • Unless safety insuperable problem clinical trials of epo gene therapy with a few years • Athletes will then be tempted to hike up hematocrit and hence endurance with single injection • Risks include blood thickening when more red blood cells present = increased risk for high blood pressure and stroke • Evidence from family’s mutation where father died in his 50s of stroke, son had heart attack at 40 (Josef Prachal, University Alabama, Birmingham) P4 Secret weapon

  17. Clotting topic 1 More problems with gene therapy • Once gene inserted it cannot be turned off • Some monkeys in experiment made too much epo • Had to be bled to thin blood and keep them alive • Athletes might also need frequent bleeding to keep hematocrit low and prevent strokes • However high blood pressure and atherosclerosis would remain a risk (Prchal) • Goldspink suggests another sort of gene therapy could build muscles P4 Secret weapon

  18. Text book reference 6.5 mRNA splicing Insulin-like growth factor (igf-1) • Hard exercise leaving you ‘sore’ build muscles because ‘micro-tears’ occur in muscle fibres • Repair involves fibres being strengthened with extra proteins • A protein IGF-1 is turned on by stretch or exercise over-load and plays a part in repair process (IGF- 1 plays many roles in the body, produced by liver in response to growth hormone) • A single gene produces five different forms of IGF-1 due to the way it is spliced. P4 Secret weapon

  19. Pumping Genes • Goldspink (Royal Free, London) working on gene therapy for muscular dystrophy • Mechano growth factor (MGF) is a form of IGF-1 made in muscle tissue, does not circulate in blood • Injected mice muscle with MGF gene, muscle grew by 20% in 2 weeks – “we seem to have found the magic potion that makes muscles grow” • Sweeney (Pennsylvania) similar results with a different IGF-1 made in liver and muscle • In blood it raises blood sugar level, but in muscle repairs and builds them P4 Pumping genes

  20. Sweeney et al • Used adenovirus to deliver IGF-1 gene to mice leg muscles • Even without exercise muscles had grown 15% in 3 months • Bodybuilders very interested, people could custom-build their physiques/re-engineer body • Could be ‘muscle men’ naturally express much more IGF-1 genes than ‘weaklings’ • Quite safe as protein produced stays in muscle and does not circulate • Therefore if injected into biceps will not lead to enlarged heart or raised blood sugar levels P4/5 Pumping genes

  21. Topic 4 drug testing Athletes and IGF-1 gene therapy • Very attractive to athletes • Build muscle by 20% easily, could be up to 50% with other growth factors • IGF-1 gene therapy could be available as soon as 2 years time • Rosenthal (geneticist, Massachusetts) warns • Mice are not humans • Different protocol would be necessary for larger animals because harder to access inside large muscles • Experimental protocol = a detailed plan of a scientific experiment that specifies experimental methods, data collection and sampling schedules P5 Pumping genes

  22. Text book reference 6.3 Body’s response to infection IGF-1 gene therapy • No guarantee how long it would work for in active athlete • Damage to muscle may cause loss of injected genes • We do not really know the turn-over rate of muscle cells • Every heart muscle cell lasts for your whole life, is the same true for skeletal muscles? • A second dose of IGF-1 gene therapy may not work as well as first • Body could build antibodies to virus vector • However using different virus vectors could circumvent this • Determined cheats will not be put off P5 Pumping genes

  23. Catching cheats • Will authorities finally lose battle over drugs in sport? • Catlin (biochemist, Olympic testing lab) had no doubt cheats will resort to gene doping “I don’t like what they do – its dirty – but I have to admit I’m impressed by the sophistication of doctors on the other side” P5 Catching cheats

  24. Catching cheats • Not easy - proteins from engineered genes look identical to natural ones • Could look for traces of virus vector by biopsy (medical test involving removal of cells of tissue for examination) at injection site, but need to know where injection occurred • Need less invasive treatment for testing for gene doping in athletes • Could look for abnormally high levels of gene’s product e.g. athlete inactive for 12 hours, test for MGF levels – if high shows gene abnormally active all the time P6 Catching cheats

  25. Catching cheats • Would athlete stay still for 12 hours • Would 12 hours be long enough? • Could work for epo gene doping • Would normally find little or no epo in blood • Anyone with high levels would suspect illegal doping, however may have legal Mantyranta’s mutation • Scientists will have trouble staying ahead of cheats • Yesalis – lots of money at stake and drug tests easy to circumvent • Thinks many of records in past 30 years are drug assisted P6 Catching cheats

  26. Muscle growth • Training increases muscle size but must be continued to maintain size • However researchers have discovered how muscles build up and break down and are close to creating a drug to stop body dismantling unused muscles • For use with weakness in sick and elderly/ long space flights • Would be used by ‘couch potatoes’ to stay in shape and sports cheats P6 Catching cheats

  27. Muscle growth • Idle muscle is unnecessary metabolic expense so built up muscles break down to conserve resources • Normally do not notice balance of muscle build up or break down if diet and exercise regime static • However after injury to bones or muscles or nerve supply, or starvation balance shifts and muscle breakdown obvious • People confined to bed or astronauts in microgravity have serious muscle-wasting (atrophy) problem P6 Catching cheats

  28. Atrophy • Also symptom of • Kidney failure • Cancer • AIDS • Vicious cycle develops – less muscle = less able to exercise = more atrophy (positive feedback) P6 Catching cheats

  29. Atrophy • Despite 30yrs+ research only way to prevent muscle loss is weight-bearing physiotherapy • Little use to sick and elderly • Could anabolic steroids help? • Have huge range of effects in addition to muscle growth • Some undesirable • Only seem to work in conjunction with exercise • Can we find treatment to help patients until well enough to walk, or astronauts until reach destination? P7 Catching cheats

  30. Active atrophy • Goldberg (Harvard) has been studying atrophy since late 1960s • Series of discoveries in 80s and 90s mean we now know how muscles grow and shrink • Muscle wasting is an active process controlled by a complex genetic pathway – NOT a passive side-effect of disuse or disease • If we could discover what turns this on, should be able to discover how to turn it off • Same biochemical programme is responsible what ever the cause of muscle wasting (disuse, metabolic disease or fasting) P7 Active atrophy

  31. Topic 2: amino acids, peptide bonds, proteases Ubiquitin-proteasome pathway (UPP) • Breaks down unwanted protein in cells • Once activated • Ubiquitin “destroy me” labels added to muscle proteins • Tagged proteins fed into proteasome (barrel-shaped multiprotein complex) which chops proteins down to amino acids P7 Active atrophy

  32. upp • Number of muscle filaments decrease • Number of muscle cells remains the same • They just become thinner and weaker • At least 90 genes involved – Goldberg calls them ‘atrogenes’ • Not known which atrogenes trigger atrophy however atrogin1 and muRF1 described in 2001 are essential and are the only two active during muscle atrophy • Code for ubiquitinligases – enzymes attaching “destroy me” labels to proteins

  33. atrogin1 and muRF1 • Barely active in normal muscle • Expression shoots up in sick animals • Knock out either atrogin1 or muRF1 and muscle-wasting practically stops • Results supported by Glass (at US pharmaceutical company) who discovered same two genes • Confusingly called atrogin1 -MAFbx! • Also found if genes knocked out in rats they suffered less atrophy after disuse and disease • More atrogenes found every year • A group at Purdue university has also found gene switch for muscle atrophy, and existing drug could switch it off P7 Active atrophy

  34. Text book reference 7.3 ECG 8.1 Nerve impulse Gym in a bottle • Pond and Hannon (Purdue University) found activity of gene erg1 increases in mice when muscles atrophy • Erg1 codes for potassium channel protein in cardiac and skeletal muscle tissue • Heart muscle potassium channel proteins have 2 variants - erg1a and erg1b • Allow muscle to repolarise after each beat so heart keeps its rhythm • Mutated erg1 gene cause ‘long QT’ syndrome – heart muscle cannot repolarise fast enough, can lead to sudden death SYNDROME = a group of symptoms that together are characteristic of a specific disorder, disease, or the like. P8 Gym in a bottle

  35. What does an ECG trace show us? • P wave – depolarisation of atria, leading to atrial systole • PR interval – time taken for impulse to be conducted from SAN across atria to the ventricles, through the AVN AM P8 Gym in a bottle

  36. What does an ECG trace show us? • QRS complex – wave of depolarisation resulting in ventricular systole • T wave – repolarisation (recovery) of ventricles during diastole • Atrial repolarisation is hidden by QRS complex and is small AM P8 Gym in a bottle

  37. Purdue team • Erg1a stimulates skeletal muscle atrophy • Found high levels of expression in wasting muscles due to cancer or disuse • If artificially increased expression in mice muscle cells they could induce atrophy (animal rights in experimentation topic 8 ) • Erg1b did not trigger atrophy • Existing drug (antihistamine - astemizole) blocks erg1a channels • Given to mice it completely prevented atrophy in unused muscles • Even built new muscles in normally active mice P8 Gym in a bottle

  38. Purdue team • Think erg1a protein stimulates ubiquitin-proteasome pathway (not sure how) • Astemizole could be used to erg1a channels to prevent muscle wasting HOWEVER it also blocks erg1a channels in the heart potentially causing long QT syndrome • Astemizoles were withdrawn in 1999 • Researchers must target erg1a in skeletal muscles without blocking erg1a & b channels in the heart P8 Gym in a bottle

  39. Purdue team • Pond believes this is possible because erg1a and b differ slightly at one end of protein chain • If they can find the difference they might be able to target it • Also investigating blocking erg1a expression using RNA-interference P8 Gym in a bottle

  40. Text book references Topic 3.3: lacoperon Topic 7.6 Transcription factors Foxo • Goldberg and Regeneron have focused on protein transcription factors • “Transcription factors ...turn other genes on or off” • Foxo controls the activity of many other atrogenes. • Disabling Foxo blocks atrophy and could be target for future therapies

  41. More to understand • We know insulin and insulin-like growth factor (IGF-1) are involved in muscle synthesis • They also seem to prevent atrophy by suppressing Foxo and turning off atrogin1 gene • Boosting IGF-1 levels in mice increases their strength, even with normal activity levels • This is why insulin and IGF-1 are banned in sport • Foxo is normally suppressed by insulin and IGF-1 in muscles, how does disease or inactivity activate Foxo? P8 Gym in a bottle

  42. More to understand • Pond thinks Foxo may be involved in erg1a-mediated atrophy • Erg1a does bind to transcription factors like Foxo so erg1a might trigger atrophy by interaction with Foxo • Several companies are also looking for drugs to block atrogin1 protein • Goldberg’s team looking into whether proteasome inhibitors (e.g. Velcade for cancer) might slow down muscle breakdown P8 Gym in a bottle

  43. A different approach • Wyeth are conducting trials of antibody therapy to stimulate muscle growth in people with muscular dystrophy – rather than prevent atrophy (see slide ‘Pump up the volume to understand how this might work) • Different approached have same end result and pathways could turn out to be linked P9 Gym in a bottle

  44. Valid reasons for Anti-wasting treatments ( A safer alternative to steroids) • No longer any doubt these treatments can be developed • Such anti-wasting treatments could: • Prevent muscle loss for patients confined to bed for more than a few days • Prevent wasting of diaphragm for those on ventilators • Disease need no longer lead to weakness • Broken bones would not need physiotherapy to rebuild muscles • Prevent older people becoming frail enabling them to keep on their feet and live independently for longer P9 Gym in a bottle

  45. Nasa • Mission to Mars • At present assume astronauts would lose 25% of muscle mass on journey to Red Planet • On arrival would be too weak to walk, let alone put on space suit and carry out repairs • Hence Goldberg’s work is funded by NASAs National Space Bioremedial Research Institute, Houston, Texas P9 Gym in a bottle

  46. Anti-wasting drugs and cheats • Goldberg’s work is for medical and space applications however it will also be tempting to cheats and couch potatoes • Muscle size is not everything – endurance training produces physiological changes • Better blood supply to muscles • More mitochondria in muscle cells • Drugs to maintain muscle size will not • Keep you fit • Give any of the benefits of exercise like • Stronger bones • ‘smarter brains’ P9 Gym in a bottle

  47. Anti-wasting drugs and cheats • More muscle does burn extra calories • Will keep you stronger if you miss the gym • May encourage people to exercise more rather than less because less painful to start up again • Until the arrival of a ‘gym in a bottle’ the best way to lower Foxo and prevent muscle atrophy? • Increase IGF-1 • Stimulate insulin production • How? • Eat regularly • Do a bit of exercise !! P9 Gym in a bottle

  48. Pump up the volume • German baby 6 years ago born with double normal muscle mass and virtually no fat • At age 5 could hold 3kg in each outstretched arm • Schuelke (Paediatrician, Berlin) discovered baby had mutation in both copies of gene coding for the muscle growth inhibitor myostatin • His mother, a former sprinter, has a mutation in one copy • Extended family reported to have unusual strength • Baby is first known individual to have mutations in both copies P9 Pump up the volume

  49. Blocking myostatin • Mice with blocked myostatin grow twice as muscular as usual • Wyeth have clinical trail approval to see if blocking myostatin with antibody therapy could be another way to prevent further muscle loss in people with muscular dystrophy • Muscular dystrophy causes muscle cells to die not just atrophy as in disease or disuse • Myostatin keeps muscle stem (satellite) cells in check • Without myostatin stem cells should give rise to new muscle cells P10 Pump up the volume

  50. Muscular dystrophy • Blocking myostatin will not cure the underlying cause of muscular dystrophy but could help compensate for lost tissue • However if exhausts supply of stem cells the reprieve would only be temporary • Antibody trials under way at centres around the world, first results expected soon • Hoped myostatin blockers will treat other kinds of muscle wasting P10 Pump up the volume

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