Molecular diagnosis of muscular dystrophy. Gene therapy strategies for muscular dystrophy. Biotechnology techniques

1. Gene Therapy for
Muscular Dystrophy
By:
Mohamed Samir El-Asaly
PT, CKTP
Under Supervision:
Porf. Dr.: Mokhtar M. ElZawahri

2. Introduction
 The first historical account of
muscular dystrophy appeared in
1830, when Sir Charles Bell wrote
an essay about an illness that
caused progressive weakness in
boys.
 Six years later, another scientist
reported on two brothers who
developed generalized weakness,
muscle damage, and replacement of
damaged muscle tissue with fat and
connective tissue. At that time the
symptoms were thought to be signs
of tuberculosis.

3. Introduction
 In the 1850s, descriptions of boys
who grew progressively weaker, lost
the ability to walk, and died at an
early age became more prominent in
medical journals.
 In the following decade, French
neurologist Guillaume Duchenne gave
a comprehensive account of 13 boys
with the most common and severe
form of the disease. It soon became
evident that the disease had more
than one form, and that these
diseases affected people of either sex
and of all ages.

4. What is muscular
Dystrophy
 Muscular dystrophy is a group of diseases that cause
progressive weakness and loss of muscle mass.
 In muscular dystrophy, abnormal genes (mutations)
interfere with the production of proteins needed to
form healthy muscle.
 There are many different kinds of muscular
dystrophy. Symptoms of the most common variety
begin in childhood, primarily in boys.

5. What is muscular
Dystrophy
 Some people who have muscular dystrophy will
eventually lose the ability to walk. Some may
have trouble breathing or swallowing.
 There is no cure for muscular dystrophy. But
medications and therapy can help manage
symptoms and slow the course of the disease.

6. Anatomy of muscle fibers

7. What is dystrophin?
 Dystrophin is a protein located
between the sarcolemma and
the outermost layer of
myofilaments in the muscle fiber
(myofiber).
 Sacrolemma also called the
myolemma, is cell membrane
of a striated muscle fibers.
 Dystrophin supports muscle fiber
strength, and the absence of
dystrophin reduces muscle
stiffness, increases sarcolemmal
deformability and helps to
prevent muscle fiber injury.

8. What is dystrophin??
 Dystrophin is part of an incredibly complex group of
proteins that allow muscles to work correctly. The
protein helps anchor various components within
muscle cells together and links them all to the
sarcolemma - the outer membrane.
 If dystrophin is absent or deformed, this process
does not work correctly and disruptions occur in the
outer membrane. This weakens the muscles and can
also actively damage the muscle cells themselves.

9. Dystrophin gene
 The gene for dystrophin
production sits on the X
chromosome. If a normal
gene for dystrophin is present,
then the protein will be made.
 If the gene is missing or
altered, dystrophin may not
be produced at all or only in
abnormal forms, resulting in
Duchenne muscular
dystrophy.

11.  Eukaryote pre-mRNAs often have intervening introns that must be
removed during RNA processing (as do some viruses).
intron = non-coding DNA sequences between exons in a gene.
exon = expressed DNA sequences in a gene, code for amino acids.
1993: Richard Roberts (New England Biolabs) & Phillip Sharp (MIT)
Introns and exons

12. Dystrophin gene biology
 The X-linked dystrophin gene (DMD) is by far the largest
of the 19,000 genes that encode proteins in the human
genome.
 Its 79 exons cover 2.6 million base pairs (bp).
 This large size makes the gene prone to rearrangement
and recombination events that cause mutations.
 In most cases, the mutations are deletions of one or more
exons.
 In general mutations that disrupt the reading frame of the
dystrophin transcript and lead to prematurely aborted
dystrophin synthesis cause Duchenne muscular dystrophy
(DMD).

13. Dystrophin gene

14. Types of
Muscular
Dystrophy
Duchenne
Becker MD
Congenital MD
Emery-Dreifuss
MD
Distal MD
Facioscapulohumera
l MD (FSHD)
Myotonic
dystrophy
(DM1)
Limb-girdle MD
(LGMD)
Oculopharynge
al MD (OPMD)

15. Duchenne MD
 Is the most common childhood form of
MD, accounting for approximately 50
percent of all cases. Because
inheritance is X-linked recessive (caused
by a mutation on the X, or sex
chromosome)
 Duchenne MD primarily affects boys,
although girls and women who carry the
defective gene may show some
symptoms.

16. Duchenne Muscular
Dystrophy Inheritance
 DMD is inherited in an
recessive pattern (defect at
Xp21 locus)
 Females will typically be
carriers for the disease while
males will be affected
 The son of a carrier mother
has a 50% chance of
inheriting the defective gene
from his mother.
 The daughter of a carrier
mother has a 50% chance of
being a carrier or having two
normal copies of the gene

17. Prevalence of DMD
 Affects one in 3500 to 5000 newborn males
 1/3 of these with previous family history
 2/3 sporadic
 There are two groups that make up the
sporadic cases. ½ of the sporadic cases, there
was a genetic mutation in the mother’s egg or
a genetic mutation early in embryo
development that led to the condition.
 In the other ½ of sporadic cases, the mother is
a carrier, but she is carrying a new mutation
that occurred in either her mother’s egg, her
father’s sperm, or early in her development.
 This explains the cases where a boy is born
with Duchenne muscular dystrophy into a
family with absolutely no history of the
condition

18. Initial Symptoms of DMD
 Delayed developmental milestones
 A waddling gait
 Pain and stiffness in the muscles
 Difficulty with running and jumping
 Walking on toes
 Particularly large calf muscles
 Difficulty sitting up or standing
 Learning disabilities, such as
developing speech later than usual
 Frequent falls.

19. Later symptoms of DMD
 Inability to walk
 A shortening of muscles and tendons, further limiting
movement.
 Breathing problems can become so severe that assisted
breathing is necessary.
 Curvature of the spine can be caused if muscles are not strong
enough to support its structure.
 The muscles of the heart can be weakened, leading to cardiac
problems.
 Difficulty swallowing; this can cause aspiration pneumonia and a
feeding tube is sometimes necessary.

20. Signs of DMD
 Has a hard time lifting his head or has a
weak neck
 Has a hard time walking, running, or
climbing stairs
 Is not speaking as well as other kids his
age
 Needs help getting up from the floor or
walks his hands up his legs in order to
stand (see Gower Maneuver, right)
 Has calves that look bigger than normal
(pseudohypertophy)
 Walks on his toes and waddles
 Walks with his chest pointed out.

21. Becker MD
 Is less severe than but closely related to
Duchenne MD.
 People with Becker MD have partial but
insufficient function of the protein dystrophin.
 There is greater variability in the clinical course
of Becker MD compared to Duchenne MD.
 The disorder usually appears around age 11 but
may occur as late as age 25, and affected
individuals generally live into middle age or
later.

22. Becker MD
 The rate of progressive, symmetric (on both
sides of the body) muscle atrophy and
weakness varies greatly among affected
individuals.
 Many individuals are able to walk until they are
in their mid-thirties or later, while others are
unable to walk past their teens.
 Some affected individuals never need to use a
wheelchair. As in Duchenne MD, muscle
weakness in Becker MD is typically noticed first
in the upper arms and shoulders, upper legs,
and pelvis.

23. Symptoms Becker MD
Early symptoms of Becker MD include:
 walking on one's toes.
 Frequent falls, and difficulty rising from the
floor.
 Calf muscles may appear large and healthy
as deteriorating muscle fibers are replaced
by fat, and muscle activity may cause
cramps in some people.
 Cardiac complications are not as consistently
present in Becker MD compared to Duchenne
MD, but may be as severe in some cases.
 Cognitive and behavioral impairments are
not as common or severe as in Duchenne
MD, but they do occur.

24. Becker MD
 Mutations in the dystrophin gene that do not
disrupt the translational reading frame result in
the milder Becker muscular dystrophy (BMD)
phenotype, which is found in 1 in every 20,000
newborn boys.
 BMD patients therefore show intermediate to
mild phenotypes and have much longer life
expectancies.

25. How are the muscular
dystrophies diagnosed?
 Individual's medical history.
 Complete family history.
 It is also important to rule out any
muscle weakness.
 Thorough clinical and neurological
exams can rule out disorders of the
central and/or peripheral nervous
systems, identify any patterns of
muscle weakness and atrophy, test
reflex responses and coordination, and
look for contractions.

26. Muscular Dystrophy
Diagnosis
 Exercise tests
 Nerve conduction velocity studies
 Electromyography (EMG)
 Electron microscopy
 Muscle biopsies

27. Blood and urine tests
 Creatine kinase is an enzyme that leaks out of
damaged muscle.
 Elevated creatine kinase levels may indicate
muscle damage, including some forms of MD,
before physical symptoms become apparent.
 Levels are significantly increased in the early
stages of Duchenne and Becker MD.
 Testing can also determine if a young woman is
a carrier of the disorder.

28. Blood and urine tests
 Myoglobin is measured when injury or
disease in skeletal muscle is suspected.
 Myoglobin is an oxygen-binding protein
found in cardiac and skeletal muscle
cells.
 High blood levels of myoglobin are found
in people with MD.

29. Blood and urine tests
 The level of serum aldolase, an enzyme
involved in the breakdown of glucose, is
measured to confirm a diagnosis of
skeletal muscle disease.
 High levels of the enzyme, which is
present in most body tissues, are noted
in people with MD and some forms of
myopathy.

30. Diagnostic imaging
 Magnetic Resonance
Imaging (MRI), is used to
examine muscle quality,
any atrophy or
abnormalities in size, and
fatty replacement of
muscle tissue, as well as to
monitor disease
progression.
 Ultrasound imaging (also
known as sonography),
Ultrasound may be used to
measure muscle bulk.

31. Immunofluorescence
 Testing can detect specific
proteins such as dystrophin
within muscle fibers. Following
biopsy, fluorescent markers are
used to stain the sample that
has the protein of interest.
 In this example: Dystrophin
IMF
1. Normal: Localized to myocyte
membrane.
2. BMD: Present but reduced.
3. DMD: Completely absent

32. Genetic counseling
 Two tests can be used to help expectant parents find out if
their child is affected.
1. Amniocentesis, done usually at 14-16 weeks of pregnancy,
tests a sample of the amniotic fluid in the womb for genetic
defects (the fluid and the fetus have the same DNA). Under
local anesthesia, a thin needle is inserted through the
woman's abdomen and into the womb. About 20 milliliters
of fluid (roughly 4 teaspoons) is withdrawn and sent to a lab
for evaluation. Test results often take 1-2 weeks.

33. Genetic counseling
2. Chorionic villus sampling, or CVS, involves the removal and
testing of a very small sample of the placenta during early
pregnancy. The sample, which contains the same DNA as
the fetus, is removed by catheter or a fine needle inserted
through the cervix or by a fine needle inserted through the
abdomen. The tissue is tested for genetic changes
identified in an affected family member. Results are usually
available within 2 weeks.

34. Multiplex PCR
 The most basic method
still in regular use
involves multiplex PCR of
the exons known to be
most commonly deleted.
 This method was first
published by
Chamberlain et al. in
1988.

35.  Analysis of dystrophin gene by multiplex PCR. I:
patient with deletion of exon 45, II: patient with
deletion of exon 48, and III: normal control.

36. Multiplex PCR
 The advantage of this method is its
relative simplicity.
 However it does not detect
duplications, does not characterize
all deletion breakpoints, and cannot
be used for carrier testing of
females.

37. Quantitative analysis
 Quantitative analysis of all exons of the gene
have brought about an improvement in
mutation detection rate, as they will detect all
exon scale deletions as well as duplications.
 Able to detect mutations in carrier females.
 Of the quantitative methods available, multiplex
ligation-dependent probe amplification (MLPA –
a commercial kit developed by MRC-Holland) is
now the most widely used.

38. Quantitative analysis
 Use of oligonucleotide-based array comparative
genomic hybridisation (array-CGH).
 This method analyses copy number variation
across the entire gene.
 Has the added advantages of detecting complex
rearrangements and large scale intronic
alterations and delineating mutation break-
points much more closely.

39. Full Sequence Analysis
 If no deletion or duplication is detected, then, in
the case of DMD patients, full sequence analysis
should be undertaken.
 Sequencing can be carried out on either
genomic DNA or muscle-derived cDNA.
 Analysis of genomic DNA has the advantage
that it does not require the patient to undergo a
muscle biopsy.

40. Full Sequence Analysis
 Analysis of genomic DNA will not detect
mutations in the 2% of cases with
complex rearrangements or deep intronic
changes.
 Analysis of muscle RNA therefore has a
slightly higher sensitivity, and is more
amenable to laboratories with less
automation, however the requirement for
a muscle biopsy is a drawback.

41. Treatments for DMD
To improve
breathing:
 O2 therapy
 Ventilator
 Scoliosis surgery
 Tracheotomy

42. Treatments for DMD
 To improve mobility:
 Physical therapy
 Surgery on tight joints
 Prednisone
 Non-steroidal medications
 Wheelchair

43. Dystrophin gene
 Dystrophin consists of 4 main
domain:
1. The N-terminal domain (red)
binds to F-ACTIN.
2. The cysteine-rich domain (green)
binds to β-DYSTROGLYCAN (β-
DG).
3. The C-terminal domain (yellow)
binds to DYSTROBREVINS and
SYNTROPHINS.
4. The central coiled-coil rod domain
(blue) contains 24 SPECTRIN-like
repeats (R1–R24) and 4 ‘hinge’
regions

44. Mini- and Micro-
dystrophins
 Reduction of the dystrophin transgene size.
 A large range of deletions in dystrophin cause
only mild phenotypes in BMD patients.
 So, large parts of the gene seem not to be vital
for function.
 To map the regions that are crucial for
dystrophin function, several transgenic mice
were engineered to carry different deletions
throughout the four dystrophin domains.

45. Mini- and Micro-
dystrophins
 Deletions in the N-terminal domain were associated with
relatively mild phenotypes, which indicates that this
region might be important but not essential for
attachment to actin and the cytoskeleton.
 By contrast, deletions in the cysteine-rich domain cause
severe dystrophy, owing to disruption of the entire
dystrophin–glycoprotein complex.
 The C-terminal domain, with its various alternative
splicing patterns, seems not to be required for the
assembly of this complex.
 A series of large deletions that were evaluated in the
central rod domain indicated that although the rod
structure is indispensable, the number of repeats can be
markedly reduced.

46. Mini-dystrophins
 A 6.2 kb mini-construct that contained 8 repeats and
hinge regions 1, 3 and 4, was engineered to mimic the
exon 17–48 deletion in a BMD patient described by
England and colleagues.
 This construct was found to be completely functional:
transgenic mice that carried this construct showed non-
dystrophic muscle morphology and normal force
generation.

47. Micro-dystrophins
 Further reduction is feasible, as shown by several micro-
constructs (3.6–4.2 kb) that were highly effective in
supporting almost normal muscle structure and function,
at least in mice.
 The smallest effective micro-construct was only 3.6 kb in
size and carried 4 repeats and hinge regions 1, 2 and 4.

48. Gene Therapy Strategies
 Gene augmentation approaches deliver
functional cDNA, such as micro- dystrophin or
micro-utrophin cDNA, to compensate the
function of dystrophin protein.
 Retroviral vectors or DNA transposonvectors
stably integrate the therapeutic cDNA into
chromosomes randomly.
 Due to the large size of the dystrophin or
utrophin cDNA, transduction efficiency is one of
the biggest obstacles.

49. Gene Therapy
Strategies
 Genome editing approaches modify the mutated
gene specifically, but off-target mutagenesis is a
concern. The delivery of programmable nucleases is
unexplored in the context of muscle tissue.
 Exon skipping uses antisense oligonucleotides to
modulate the splicing patterns of a particular exon.
Systematic delivery is feasible for antisense
oligonucleotides, but the effect is transient.
 Risk of off-target effects or posttranslational
suppression of the target gene should also be
considered.

51. Gene Replacement
Therapy
 To replace a defective dystrophin gene, an artificial
dystrophin cDNA construct must be transferred into the
nuclei of muscle cells, where it must be expressed and
regulated appropriately.
 So, to deliver the 14 kb dystrophin cDNA vectors with a
large capacity were needed.
 The capacity of first generation adenoviral vectors (up to
8 kb) was too small.
 Later, high capacity (28 kb) ‘gutless’ vectors, from which
all adenoviral genes had been removed, bypassed this
restriction and delivered extra benefits in the form of
reduced host immune response to the viral vector and
improved persistence of transgene expression in muscle.

52. Gene Replacement
Therapy
 However, two crucial problems need to be
overcome before adenoviral vectors can be used
therapeutically:
1. They are too large to easily cross the
EXTRACELLULAR MATRIX that surrounds
mature myofibres
2. There are not many adenoviral attachment
receptors on the surface of myofibres

53. Gene Replacement
Therapy
 In contrast to adenoviral vectors,
herpes simplex virus type-1
(HSV-1) vectors can naturally
carry large inserts.
 HSV-1 vectors have shown
relatively high TRANSDUCTION
levels in vivo, but, similar to
gutless adenoviral vectors, this
is only seen in newborn and
regenerating muscle.
 The IMMUNOGENICITY and
CYTOTOXICITY of HSV-1
hampers the long-term
expression of transgenes.

55. Gene Replacement
Therapy
 The size of the full-length dystrophin cDNA is
not a problem for non-viral DNA plasmid vectors
that can be engineered to contain large inserts.
 These vectors are synthetic and non-infectious,
so they are highly suitable for use.
 This delivery strategy is inefficient in muscle
tissue, so other strategies are needed to
enhance TRANSFECTION efficiencies. Less
invasive, and preferably systemic, methods are
needed before plasmid vectors can be used.

56. Transgene delivery
with rAAV vectors
 Transgenes (for example, the mini- or micro-
dystrophins) are cloned in between the AAV
inverted terminal repeat (ITR) sequences, under
the control of a promoter of choice (for
example: CMV, MCK or CK6).

57. Transgene delivery
with rAAV vectors
 Different mini- and micro-dystrophin gene constructs were
cloned into rAAV type-2 vectors and tested in md mice.
 These studies showed that the rAAV delivery of constructs
carrying four, five or eight repeats, in combination with either
two or three hinge regions, was an effective means of treating
DMD symptoms in this model.
 Reversal of the md-associated morphological abnormalities
was observed up to at least six months post-injection,
independent of the choice of promoter (CMV, MCK or CK6), the
injected muscle (gastrocnemius or tibialis anterior) and the
age of the mice at the time of injection.
 Overall, there was widespread high expression of the mini-
and micro-dystrophins, with correct localization at the fibre
membranes and a restored dystrophin– glycoprotein complex.

58. Transgene delivery
with rAAV vectors
 The therapeutic effect was characterized by the
correction of several pathophysiological
parameters that are associated with the md
mice phenotype, such as variable fibre
diameters, myofibres with central nuclei,
reduced membrane integrity and necrosis.
 These data indicate that the rAAV-mediated
delivery of effective mini- or micro-dystrophin
gene constructs can slow or even halt the
progression of the dystrophy on a long-term
basis.

59. Transgene delivery
with rAAV vectors
 However, a greater immune response against rAAV
delivered (foreign) transgene products was observed in
dystrophic muscle compared with normal muscle. This
was attributed to the inflammatory md-muscle
environment and the effect of co-infecting antigen
presenting cells (APCs) that activate cytotoxic T
lymphocytes against the transgenic products, which
causes the destruction of transduced myofibres.
 To minimize the immune responses in rAAV-based DMD
gene-therapy studies, it will be necessary to use
immunosuppressing drugs, muscle-specific promoters to
avoid the activation of APCs and fully functional micro and
mini-dystrophin constructs to protect fibres from
degeneration and the release of neo-antigens.

60. Antisense-Induced
Exon Skipping
 Antisense oligodeoxyribonucleotide is a short
single stranded nucleic acid, typically 15-25
nucleotides in length, that has the ability to
mediate theraputic effects by directly interacting
with pre-mRNA or mRNA in a sequence specific
manner.
 Theraputic antisense are normally designed to
bind to relevant exon-intron junction in thr pre-
mRNA; blocking of splicing at the junction may
induce skipping of an adjucent exon containing
the harmful mutation.

61. Antisense-Induced
Exon Skipping
 The relatively mild BMD phenotypes that are
caused by some large deletions or nonsense
mutations have also pointed to another possible
gene-therapy strategy:
 Skipping an exon during PRE-mRNA SPLICING
to enlarge a DMD deletion so that it becomes its
nearest in-frame BMD counterpart.
 So, it was clear that skipping exons could have
some therapeutic value, but the question
remained: how could this be artificially induced?

62. Antisense-Induced
Exon Skipping

63. Antisense-Induced
Exon Skipping
 The answer came from a study on a Japanese DMD
patient, in which a 52-bp frame-disrupting deletion in
exon 19 was found to cause exon 19 skipping from the
dystrophin transcript.
 It was proposed that this region might contain an exon
recognition site (ERS) — also known as an exon-splicing
enhancer (ESE) — which is a purine-rich sequence that is
required for the correct splicing of exons with weak splice-
site consensus sequences.
 A small antisense oligodeoxyribonucleotide (ODN) was
tested and found to block this ERS sequence, as judged
from the precise skipping of exon 19 following transfection
of this ODN into human lymphoblastoid cells.

64. Antisense-Induced
Exon Skipping

65. Antisense-Induced
Exon Skipping
The skipping of only 12 different exons could theoretically correct almost 75%
of all deletions.

66. CRISPR-Cas9
 CRISPR-Cas9 is a unique technology that
enables geneticists and medical researchers to
edit parts of the genome by removing, adding
or altering sections of the DNA Sequence.
 It is currently the simplest, most versatile and
precise method of genetic manipulation and is
therefore causing a buzz in the science world.
 It can be used in a point mutation in the
dystrophin gene.

67. CRISPR-Cas9
 The CRISPR-Cas9 system consists of two key molecules
that introduce a change mutation into the DNA. These
are:
 An enzyme called Cas9. This acts as a pair of ‘molecular
scissors’ that can cut the two strands of DNA at a specific
location in the genome so that bits of DNA can then be
added or removed.
 A piece of RNA called guide RNA (gRNA). This consists of
a small piece of pre-designed RNA sequence (about 20
bases long) located within a longer RNA scaffold. The
scaffold part binds to DNA and the pre-designed sequence
‘guides’ Cas9 to the right part of the genome. This makes
sure that the Cas9 enzyme cuts at the right point in the
genome.

69. Utrophin Upregulation
 Dystrophin has a homologue called utrophin.
The utrophin gene (UTRN) maps to chromosome
6q24 and contains.
 Although the total genomic length of the
utrophin gene is only approximately one-third of
that of the dystrophin gene, its transcript is (at
13 kb) almost as large. Dystrophin and utrophin
are highly similar and one probably originated
from a duplication of the other.
 The most prominent difference is that it lacks
the spectrin-like repeats 15 and 19, and 2 hinge
regions of dystrophin.

70. Utrophin Upregulation
 Utrophin is ubiquitously expressed in most
tissues— most prominently in lungs, blood
vessels and the nervous system.
 In muscle, its local expression is
maturation dependent: in fetal muscle it
is initially dispersed over the
SARCOLEMMA, during development it is
gradually replaced by dystrophin and in
mature muscle it is located only at the
neuromuscular and myotendinous
junctions.
 At the post-synaptic membrane of the
neuromuscular junctions, utrophin co-
localizes with the ACETYLCHOLINE
receptors and is thought to have a
structural and functional role in the
differentiation and maintenance of
postsynaptic membrane domains.

71. Utrophin Upregulation
 By contrast, in the regenerating muscle of DMD
patients, mdx mice and dystrophin-deficient cats,
utrophin was found to be both upregulated and
redistributed to the sarcolemma.
 This latter observation led to the hypothesis that
utrophin might have a complementary, as well as a
protective, role in dystrophic muscle.
 Further support for this hypothesis comes from
utrophin– dystrophin double-deficient mice, which
show severe progressive muscle weakness,
neuromuscular and myotendinous-junction
abnormalities, and die prematurely.

72. Utrophin Upregulation
Utrophin to treat dystrophin deficiency:
 Studies in mdx mice showed the feasibility of utrophin
upregulation to treat DMD.
 High expression of a truncated utrophin transgene at the
sarcolemma notably reduced dystrophic pathology and
intracellular calcium homeostasis, and improved
mechanical muscle performance.
 Furthermore, adenoviral delivery of mini-utrophin restored
the dystrophin–glycoprotein complex, reduced the number
of centrally nucleated fibres and so rescued the dystrophic
phenotypes of three animal models of DMD.
 The expression of full-length utrophin mRNA in transgenic
mdx mice produced even better results, despite
expression levels that were at most 50% of the normal
endogenous levels.

73. MYOBLAST
TRANSPLANTATION
 Is another dystrophin gene delivery strategy, also has
problems that have prevented its use in the clinic:
specifically immune rejection, limited cell spreading and
poor survival of the myoblasts immediately post-
transplantation.
 Recently, similar cell-therapy strategies, which use stem
cells that are derived from bone marrow, muscle or blood
vessels, have had notable successes in dystrophic mouse
models and DMD muscle.

74. Ex vivo gene therapy
 Ex vivo gene therapy approaches using iPS cells
Induced pluripotent stem cells. A scheme for
iPS cell-mediated ex vivo gene therapy
approaches for DMD.
 Skin fibroblasts or monocytes from peripheral
blood are reprogrammed to iPS cells by
transient expression of the Yamanaka factors.
 The dystrophin mutation can then be repaired
using genome engineering technologies. Such
corrected iPS cells can be further differentiated
into myoblasts to form myofibers.

75. Ex vivo gene therapy
 Either myoblasts or myofibers can be
transplanted to patients, but only for transient
recovery, as myoblasts or myofibers will
eventually die after cellular turnover.
 An ideal approach would be to differentiate iPS
cells into satellite cells, which are muscle stem
cells, to gain long-term self-renewal and
regeneration capacity in the myofibers.
 Currently, ex vivo expansion of primary satellite
and genome editing is challenging, but progress
here could circumvent the use of iPS cells.

77. The future of DMD
therapy
 Steady progress in understanding the gene and
its function has pointed to several innovative
therapeutic strategies.
 It now seems reasonable to expect that the next
decade will see great advances in this field.
 Considering its efficiency and relative simplicity,
the antisense approach seems the next
candidate (and probably the most promising so
far) for clinical trials.