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UNIT 5 PRE-RELEASEScientific article
2011
1
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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
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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
Text book reference 7.6
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)
Text book reference 7.6
Action of hormones
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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 longerstimulated
.and no extra red blood cells are produced
Text book reference 7.6
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)
Text book reference 7.6
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
Text book reference 8.7
genetic modification
Text book reference 7.6
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
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Text book reference 7.6
Performance-enhancing
substances
Text book reference 7.2
oxygen required for ATPproduction in respiration
Text 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
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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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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Text book reference 6.3
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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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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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
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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
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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
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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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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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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 them19
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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
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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
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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
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Text book reference 6.3
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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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
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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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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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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
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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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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
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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
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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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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
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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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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
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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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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
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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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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
40
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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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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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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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
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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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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
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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
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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
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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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