UNIT 5 PRE-RELEASE

1 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
1. Gene-doping to grow bigger muscles
2. Erythropoietin to increase oxygen carrying-capacity of
the blood

P2 Muscles, genes and gym in a bottle

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

 Text book reference 7.6 –
Action of hormones
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 4

P2 Muscles, genes and gym in a bottle

 Text book reference 7.6 –
Action of hormones
ACTION OF ERYTHROPOIETIN
Hormone receptor
for erythropoietin
Hormone -
erythropoietin
on red blood cell precursor in
bone marrow

Text book reference 7.6

transcription factors

activates transcription factors leading to

mRNA and translation to proteins which cause 5
increase in red blood cell manufacture (also
inhibits apoptosis)
P2 Muscles, genes and gym in a bottle
 Text book reference 7.6 –
negative feedback

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

P2 Muscles, genes and gym in a bottle

 Text book reference 7.6 –
Action of hormones
WHAT MUTATION DID MANTYRANTA HAVE?
Hormone receptor
for erythropoietin
Hormone -
erythropoietin
 Mantyranta’s mutation
on red blood cell
meant his EpoR precursor in bone
marrow
receptor was never
turned off so he
continually made new
red blood cells
 This is a very rare
mutation

activates transcription factors leading to

mRNA and translation to proteins which cause 7
increase in red blood cell manufacture (also
inhibits apoptosis)
P2 Muscles, genes and gym in a bottle
 Text book reference 8.7 –
genetic modification

Text book reference 7.6

EPO DOPING Performance-enhancing
substances

 Injecting epo means anyone can increase their red blood

cells
 1989 Biotechnology company Amgen began marketing a
form of epo produced by recombinant bacteria for
treating severe anaemia from suffered by AIDS and
kidney patients
 It then began to be exploited by athletes

 Employee of Festina cycling team found with car load of

performance-enhancing drugs including epo at 1998
Tour de France
 Swiss rider Alex Zulle ‘Doping is part of the business of
8
cycling’
P2 Muscles, genes and gym in a bottle
 Text book reference 7.6
Performance-enhancing
substances

SECRET WEAPON Text book reference 7.2

oxygen required for ATP
production in respiration
 Widespread doping? Text book reference 7.1 ATP
 Australian required for muscle contraction
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 2.7/8.7
genetic engineering with
viruses

‘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

10

P3 Secret weapon
 Text book reference 6.3
Body’s response to infection

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?

11

P3 Secret weapon
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

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P3 Secret weapon
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

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P3 Secret weapon
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
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 Injection of recombinant erythropoetin

P3 Secret weapon
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
15

P3 Secret weapon
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)
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P4 Secret weapon
 Clotting topic 1

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

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P4 Secret weapon
 Text book reference 6.5 mRNA
splicing
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.

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P4 Secret weapon
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 them
19

P4 Pumping genes
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
20
heart or raised blood sugar levels
P4/5 Pumping genes
 Topic 4 drug testing

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 21

P5 Pumping genes
 Text book reference 6.3 Body’s
response to infection

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
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P5 Pumping genes
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”

23

P5 Catching cheats
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
24

P6 Catching cheats
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 assisted
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P6 Catching cheats
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

26

P6 Catching cheats
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

27

P6 Catching cheats
ATROPHY
 Also symptom of
 Kidney failure
 Cancer
 AIDS

 Vicious cycle develops – less muscle = less able to

exercise = more atrophy (positive feedback)

28

P6 Catching cheats
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
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
30
or fasting)
P7 Active atrophy
 Topic 2: amino acids, peptide
bonds, proteases

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

31

P7 Active atrophy
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
32
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

33
muscle atrophy, and existing drug could switch it off
P7 Active atrophy
 Text book reference 7.3 ECG
8.1 Nerve impulse
GYM IN A BOTTLE
 Pond and Hannon (Purdue University) found activity of
gene erg1 increases in mice when muscles atrophy
 Erg1 codes for potassium channel protein in cardiac and
skeletal muscle tissue
 Heart muscle potassium channel proteins have 2 variants
- erg1a and erg1b
 Allow muscle to repolarise after each beat so heart keeps
its rhythm
 Mutated erg1 gene cause ‘long QT’ syndrome – heart
muscle cannot repolarise fast enough, can lead to sudden
death

SYNDROME = a group of symptoms that together are 34

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

P8 Gym in a bottle
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
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

36

AM
P8 Gym in a bottle
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
37
 Even built new muscles in normally active mice

P8 Gym in a bottle
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

38

P8 Gym in a bottle
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

39

P8 Gym in a bottle
 Text book references
Topic 3.3: lac operon
FOXO Topic 7.6 Transcription factors

 Goldberg and Regeneron have focused on protein

transcription factors
 “Transcription factors ...turn other genes on or off”

 Foxo controls the activity of many other atrogenes.

 Disabling Foxo blocks atrophy and could be target for

future therapies

40
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?

41

P8 Gym in a bottle
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

42

P8 Gym in a bottle
A DIFFERENT APPROACH

 Wyeth are conducting trials of antibody therapy to

stimulate muscle growth in people with muscular
dystrophy – rather than prevent atrophy (see slide
‘Pump up the volume to understand how this might
work)
 Different approached have same end result and pathways
could turn out to be linked

Prevent Stimulate
muscle muscle
atrophy growth

More
muscle
tissue 43

P9 Gym in a bottle
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

44

P9 Gym in a bottle
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

45

P9 Gym in a bottle
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’ 46

P9 Gym in a bottle
 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?
 IncreaseIGF-1
 Stimulate insulin production

 How?
 Eatregularly 47
 Do a bit of exercise !!
P9 Gym in a bottle
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
48

P9 Pump up the volume

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 cells
49

P10 Pump up the volume

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

50

P10 Pump up the volume