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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 - PowerPoint PPT Presentation
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UNIT 5 PRE-RELEASEScientific article2011
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MUSCLES, GENES AND GYM IN A BOTTLE Gene therapies to treat genetic disease can
be abused by athletes1. Gene-doping to grow bigger muscles2. Erythropoietin to increase oxygen carrying-
capacity of the blood
P2 Muscles, genes and gym in a bottle
Text book reference 8.7 – genetic modification
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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
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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
Text book reference 7.6 – Action of hormones
P2 Muscles, genes and gym in a bottle
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ACTION OF ERYTHROPOIETIN
Hormone - erythropoietin
Hormone receptor for erythropoietin
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)
Text book reference 7.6 – Action of hormones
P2 Muscles, genes and gym in a bottle
Text book reference 7.6 transcription factors
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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
Text book reference 7.6 – negative feedback
P2 Muscles, genes and gym in a bottle
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WHAT MUTATION DID MANTYRANTA HAVE?
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
Hormone - erythropoietin
Hormone receptor for erythropoietin
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)
Text book reference 7.6 – Action of hormones
P2 Muscles, genes and gym in a bottle
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EPO DOPING 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’
Text book reference 8.7 – genetic modification
Text book reference 7.6 Performance-enhancing substances
P2 Muscles, genes and gym in a bottle
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SECRET WEAPON 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
P3 Secret weapon
Text book reference 7.6 Performance-enhancing substancesText book reference 7.2 oxygen required for ATP production in respirationText book reference 7.1 ATP required for muscle contraction
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‘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
Text book reference 2.7/8.7 genetic engineering with viruses
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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
Text book reference 6.3 Body’s response to infection
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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
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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
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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
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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
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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
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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
Clotting topic 1
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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
Text book reference 6.5 mRNA splicing
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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 themP4 Pumping genes
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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 levelsP4/5 Pumping genes
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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
Topic 4 drug testing
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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
Text book reference 6.3 Body’s response to infection
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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
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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
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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 assistedP6 Catching cheats
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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
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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
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ATROPHY Also symptom of
Kidney failure Cancer AIDS
Vicious cycle develops – less muscle = less able to exercise = more atrophy (positive feedback)
P6 Catching cheats
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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
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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
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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
Topic 2: amino acids, peptide bonds, proteases
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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 ubiquitin ligases – enzymes attaching “destroy me” labels to proteins
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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
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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
P8 Gym in a bottle
Text book reference 7.3 ECG8.1 Nerve impulse
SYNDROME = a group of symptoms that together are characteristic of a specific disorder, disease, or the like.
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
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P8 Gym in a bottle
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
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P8 Gym in a bottle
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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 miceP8 Gym in a bottle
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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
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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
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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
Text book referencesTopic 3.3: lac operonTopic 7.6 Transcription factors
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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
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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
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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
P9 Gym in a bottle
More muscl
e tissue
Prevent muscle atrophy
Stimulate muscle growth
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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
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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 Goldberg’s work is funded by NASAs
National Space Bioremedial Research Institute, Houston, Texas
P9 Gym in a bottle
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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
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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
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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
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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 cellsP10 Pump up the volume
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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