Pre Release 2011

8/6/2019 Pre Release 2011

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UNIT 5 PRE-RELEASEScientific article

2011

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MUSCLES, GENES AND GYM IN A BOTTLE

Gene therapies to treat genetic disease can be

abused by athletes

1. Gene-doping to grow bigger muscles

2. Erythropoietin to increase oxygen carrying-capacity of the blood

2

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 tohave 25-50% more red blood cells than normal

3

..



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HOW ERYTHROPOIETIN FUNCTIONS

Erythropoietin (epo) is aglycoprotein hormone

It is produced and released bythe kidney when oxygen level ofthe blood are low

Epo then travels in the blood tothe bone marrow

It combines with the

erythropoietin receptor (EpoR)on the cell surface membrane ofred blood cell precursors causingthem to increase the number ofred blood cell produced 4


Action of hormones



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ACTION OF ERYTHROPOIETIN

5

Hormone -

erythropoietin

Hormone

receptor forerythropoietin

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)


Action of hormones



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 longerstimulated

.and no extra red blood cells are produced


negative feedback



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WHAT MUTATION DID MANTYRANTA HAVE?

Mantyrantas

mutation meant his

EpoR receptor was

never turned off sohe continually made

new red blood cells

This is a very rare

mutation

7

Hormone -

erythropoietin

Hormone

receptor forerythropoietin

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)


Action of hormones



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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 byrecombinant 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 carload of performance-enhancing drugs including

epo at 1998 Tour de France

Swiss rider Alex Zulle Doping is part of the

business of cycling8


genetic modification


Performance-enhancing

substances



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SECRET WEAPON

Widespread doping?

y Australian Open tennis

y Cross-country skiing

y

Footbally Track and field athletics

y 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 epo9

P3 Secret weapon


Performance-enhancing

substances


oxygen required for ATPproduction in respiration


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

Mantyrantas super-gene

This gene therapy is already under developmentby several academic

groups and biotech

companies for anaemia

e.g. Avigen

Use viruses as vector

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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

y Large size so can carry big genes

Disadvantagesy Easily recognised and

destroyed by immune

system

Will immune system

destroy it before the

gene is delivered?

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P3 Secret weapon


Bodys response to

infection


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GENE THERAPY WITH ADENOVIRUS

Avigen have patented adeno-associated viruses

(AAVs) for delivering epo

y Smaller than adenovirus

y

Carries a smaller loady BUT less vulnerable to attack from immune system

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P3 Secret weapon


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GENE THERAPY SUCCESS

Both viruses have shown exceptional results

y 1997 (Leiden , University of Chicago) - adenovirus to

deliver epo gene to mice and monkeys

y Injected into muscles

y Infiltrated cells

y Inserted epo gene

y Cells pumped out epo

y Mouse hematocrits (proportion of blood volume made

up of red blood cells) up from 49% - 81%, lasted over ayear

y Monkey hematocrits up 40% - 70%, lasted 12 weeks

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P3 Secret weapon


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HEMATOCRITS

Proportion of blood volume made up of red blood cells

Typically

y males.......... 40-50%

y females....... 38-45%

y 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:

y Exercise. regular aerobic exercise raises the hematocrit.

y Living at high altitude

y Injection of recombinant erythropoetin14

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)

y

Hemaocrits 38/40% to 62/75% remained for 28 weeksof study

Risk free??

No, 18 yr old patient receiving gene therapy for

rare liver complaint died after adenovirus used todeliver gene

Currently unsure what went wrong so reviewing

safety 15

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 singleinjection

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) 16

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 tokeep hematocrit low and prevent strokes

However high blood pressure and atherosclerosis

would remain a risk (Prchal)

Goldspink suggests another sort of gene therapycould build muscles

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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 repairprocess (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 wayit is spliced.

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P4 Secret weapon


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 them19

P4 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 levels20

P4/5 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 as2 years time

Rosenthal (geneticist, Massachusetts) warns

y Mice are not humans

y Different protocol would be necessary for larger

animals because harder to access inside largemuscles

y Experimental protocol = a detailed plan of ascientific experiment that specifies experimentalmethods, data collection and sampling schedules 21

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 musclecells

y 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 off22

P5 Pumping genes


Bodys 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 dontlike 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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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 tissuefor 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 24

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 bloodAnyone with high levels would suspect illegal

doping, however may have legal Mantyrantasmutation

Scientists will have trouble staying ahead of

cheatsYesalis lots of money at stake and drug tests

easy to circumvent

Thinks many of records in past 30 years are drugassisted

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P6 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 tocreating 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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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 buildup 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 27

P6 Catching cheats


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ATROPHY

Also symptom of

y Kidney failure

y Cancer

y

AIDSVicious cycle develops less muscle = less able to

exercise = more atrophy (positive feedback)

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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? 29

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)30

P7 Active atrophy


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UBIQUITIN-PROTEASOME PATHWAY(UPP)

Breaks down unwanted protein in cells

Once activated

y Ubiquitin destroy me labels added to muscle proteins

y

Tagged proteins fed into proteasome (barrel-shapedmultiprotein complex) which chops proteins down to amino

acids

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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 thematrogenes

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 proteins32


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ATROGIN1AND 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 33

P7 Active atrophy


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GYM IN A BOTTLE

Pond and Hannon (Purdue University) found

activity of gene erg1 increases in mice whenmuscles atrophy

Erg1 codes for potassium channel protein in

cardiac and skeletal muscle tissue

Heart muscle potassium channel proteins have 2variants - 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

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P8 Gym in a bottle

Text book reference 7.3 ECG

8.1 Nerve impulse

SYNDROME = a group of symptoms that together are

characteristic of a specific disorder, disease, or the like.


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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 theventricles, through the AVN

AMP8 Gym in a bottle


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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 complexand 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 wastingmuscles due to cancer or disuse

If artificially increased expression in mice musclecells they could induce atrophy (animal rights inexperimentation 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 mice37

P8 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 blockserg1a 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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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 targetfor future therapies

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Text book references

Topic 3.3: lac operon

Topic 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-1in muscles, how does disease or inactivity activate

Foxo?

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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 interactionwith 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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P8 Gym in a bottle


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ADIFFERENT APPROACH

Wyeth are conducting trials of antibody therapyto 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

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P9 Gym in a bottle

Moremuscletissue

Preventmuscle

atrophy

Stimulatemuscle

growth


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VALID REASONS FORANTI-WASTING

TREATMENTS ( ASAFER ALTERNATIVE TO STEROIDS)

No longer any doubt these treatments can be

developed

Such anti-wasting treatments could:

y

Prevent muscle loss for patients confined to bed formore than a few days

y Prevent wasting of diaphragm for those on

ventilators

y Disease need no longer lead to weakness

y Broken bones would not need physiotherapy torebuild muscles

y Prevent older people becoming frail enabling them to

keep on their feet and live independently for longer44

P9 Gym in a bottle


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NASA

Mission to Mars

At present assume astronauts would lose 25% ofmuscle 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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P9 Gym in a bottle


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ANTI-WASTING DRUGS AND CHEATS

Goldbergs work is for medical and spaceapplications however it will also be tempting tocheats and couch potatoes

Muscle size is not everything

endurance training producesphysiological changes

y Better blood supply to muscles

y More mitochondria in muscle cells

Drugs to maintain muscle size will not

y Keep you fit

y Give any of the benefits of exercise like Stronger bones

smarter brains 46

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 ratherthan less because less painful to start up again

Until the arrival of a gym in a bottle the bestway to lower Foxo and prevent muscle atrophy?

y Increase IGF-1

y Stimulate insulin production How?

y Eat regularly

y Do a bit of exercise !! 47

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 babyhad 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 copies48

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 cells49



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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

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