UNIT 5 PRE-RELEASE

Scientific article 2011

Text book reference 8.7 genetic modification

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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

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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

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Text book reference 7.6 Action of hormones

ACTION OF ERYTHROPOIETIN
Hormone erythropoietin Hormone receptor for erythropoietin

Text book reference 7.6 Action of hormones

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)
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Text book reference 7.6 negative feedback

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

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WHAT MUTATION DID MANTYRANTA HAVE?

Mantyrantas mutation meant his EpoR receptor was never turned off so he continually made new red blood cells This is a very rare mutation

Hormone erythropoietin Hormone receptor for erythropoietin

Text book reference 7.6 Action of hormones

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)
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Text book reference 8.7 genetic modification 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

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Text book reference 7.6 Performance-enhancing substances Text book reference 7.2 oxygen required for ATP production in respiration

Widespread doping?
y y y y y

Australian Open tennis Cross-country skiing Football Track and field athletics Epo rumoured to make athletes run 20% faster

Text book reference 7.1 ATP required for muscle contraction

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

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Text book reference 2.7/8.7 genetic engineering with viruses

Epo needs injecting several times a week, gene therapy would give them the equivalent of Mantyrantas 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

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Text book reference 6.3 Bodys response to infection

Genes making it pathogenic removed Advantages as a vector

y

Large size so can carry big genes Easily recognised and destroyed by immune system

Disadvantages
y

Will immune system destroy it before the gene is delivered?

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Avigen have patented adeno-associated viruses (AAVs) for delivering epo

Smaller than adenovirus y Carries a smaller load y BUT less vulnerable to attack from immune system
y

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Both viruses have shown exceptional results

y y y y y y

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
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Biotech company Chiron reported similar results in 1998 using AAVs to deliver epo gene to two BABOONS (mistake in paper)
y

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

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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 familys mutation where father died in his 50s of stroke, son had heart attack at 40 (Josef Prachal, University Alabama, Birmingham)

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Clotting topic 1

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

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Text book reference INSULIN-LIKE GROWTH FACTORsplicing 6.5 mRNA (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.

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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

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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

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Topic 4 drug testing

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Text book reference 6.3 Bodys response to infection

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Will authorities finally lose battle over drugs in sport? Catlin (biochemist, Olympic testing lab) had no doubt cheats will resort to gene doping I dont like what they do its dirty but I have to admit Im impressed by the sophistication of doctors on the other side

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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 genes product e.g. athlete inactive for 12 hours, test for MGF levels if high shows gene abnormally active all the time

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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 Mantyrantas 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

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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

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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

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Also symptom of
Kidney failure y Cancer y AIDS
y

Vicious cycle develops less muscle = less able to exercise = more atrophy (positive feedback)

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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?

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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 sideeffect 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)

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Topic 2: amino acids, peptide bonds, proteases

Breaks down unwanted protein in cells Once activated

Ubiquitin destroy me labels added to muscle proteins y Tagged proteins fed into proteasome (barrel-shaped multiprotein complex) which chops proteins down to amino acids
y

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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 ubiquitin ligases enzymes attaching destroy me labels to proteins

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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

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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.
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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

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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

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Think erg1a protein stimulates ubiquitinproteasome 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

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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

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FOXO

Text book references Topic 3.3: lac operon Topic 7.6 Transcription factors

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

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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?

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Pond thinks Foxo may be involved in erg1amediated 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 Goldbergs team looking into whether proteasome inhibitors (e.g. Velcade for cancer) might slow down muscle breakdown

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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

Prevent muscle atrophy

Stimulate muscle growth

More muscle tissue

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No longer any doubt these treatments can be developed Such anti-wasting treatments could:
y y y y y

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

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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 Goldbergs work is funded by NASAs National Space Bioremedial Research Institute, Houston, Texas

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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 y Stimulate insulin production
y

How?
Eat regularly y Do a bit of exercise !!
y
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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

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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

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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

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