Unit 5 pre-release

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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

..


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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


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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 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


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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)



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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


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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

http://www.nature.com/focus/rnai/animations/animation/animation.htm

http://www.nature.com/focus/rnai/animations/animation/animation.htm

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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

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Unit 5 pre-release