The document provides an overview of muscular dystrophy, particularly focusing on Duchenne Muscular Dystrophy (DMD), a severe genetic disorder affecting muscle function, primarily caused by the absence of dystrophin protein due to mutations in the dystrophin gene. It highlights the various inheritance patterns, symptoms, and current gene therapy advancements aimed at treating DMD, including antisense techniques and exon skipping methods that could potentially restore dystrophin production in affected individuals. Despite no known cure, the document outlines therapies and ongoing clinical trials exploring the efficacy of gene therapy in managing this condition.

1. MUSCULAR DYSTROPHY
Gene Therapy
Presented to you by
Hercolanium GDeath

2. Table of Contents
 Introdution
 Types of Muscular Dystrophy
 Duchenne’s Muscular Dystrophy
 DMD In Depth
 DMD-Dystrophin relations
 Gene Therapy Possiblilities
 More Recent and Promising Gene Therapy Trials
 Developments…

3.  Muscular dystrophy refers to a group of genetic and hereditary diseases of
the muscles that are required for movement in human body. This condition
came to light when Sir Charles Bell wrote about an illness that caused
progressive weakness in boys in 1830.
 A few more cases were initially reported. At that time the symptoms were
that of tuberculosis.
 However, it was the French neurologist, Guillaume Duchenne who gave
the description of a severe form of the disease in 1861 which is named
after him. Later it was learnt that this disease has several forms and it is
known to affect the males of all age groups.
What is Muscular Dystrophy?

4. CONT.
This condition is inherited and
different inheritance patterns
cause different muscular
dystrophies. Some cases of the
disease are mild and progress
very slowly while there are
others that cause sever
weakness of the muscles and
the affected person losses the
ability to walk. Generally the
most sever form occurs in
childhood. Some children with
this condition die in childhood
while others live into adulthood
with disability.
There is no known
cure for this condition
and inactivity can
make condition worse.
Several therapies
such as physical
therapy, occupational
therapy, and speech
therapy are being
used to help
overcome the
problem.

5. CONT.
Healthy
MDIt is an X-linked recessive
inherited disorder. The gene is
located on the X chromosome.
Since women have two X
chromosomes, a mutation that
is present in one gets
compensated by the
unaffected one so they have
milder symptoms. Since men
have only one X chromosome,
one altered copy of the gene is
sufficient to cause the disorder.
The female is the carrier and
there is 50 percent chance of
passing on the mutation in
every pregnancy. The son who
inherits this gets affected. The
daughter who inherits this
becomes the carrier.

6. CONT.
According to scientists,
muscular dystrophy can
either be recessive or
dominant, depending on the
type of the disease.
The muscular dystrophies
(MD) are a group of more than
30 genetic diseases
characterized by progressive
weakness and degeneration of
the skeletal muscles that
control movement. Some
forms of MD are seen in
infancy or childhood, while
others may not appear until
middle age or later. Muscular
dystrophies in general are a
group of genetic, degenerative
diseases primarily affecting
voluntary muscles.

7. • Poor head control
• Contractures
Some Symptoms of Muscular Dystrophy

8. • Breathing very
shallow, especially
at night; a
ventilator is often
used

9. Press me
Press me
Due to the tightness of time we
shall focus on Duchenne’s
Muscular Dystrophy and to
lesser extent on Becker
Muscular Dystrophy

11. Press Me to Go Back to
The List Slide

12. Becker muscular dystrophy (BMD)
 This type is similar to DMD, which we will
talk about later, but often much less severe.
There can be significant heart involvement.
Progression - Disease progresses slowly
and with variability. Most with BMD survive
well into mid- to late adulthood.

13. Duchenne muscular dystrophy (DMD)
 Definition - One of nine types of
muscular dystrophy, a group of
genetic, degenerative diseases
primarily affecting voluntary
muscles.
 Cause - An absence of dystrophin,
a protein that helps keep muscle
cells intact.

14. Duchenne Muscular Dystrophy
Facts
 DMD affects mostly males at a rate of 1 in 3,500 births.
 There are over 200 types of mutations that can cause any one of the
forms of muscular dystrophy.
 There are also mutations that occur within the same gene that cause other
disease types.
 DMD is the most severe and common type of muscular dystrophy.
 DMD is characterized by the wasting away of muscles.
 Diagnosis in boys usually occurs between 16 months and 8 years.
 Parents are usually the first to notice problem.
 Death from DMD usually occurs by age of 30.

15. DMD
 Onset - Early childhood - about 2 to 6 years.
 Symptoms - Generalized weakness and muscle wasting
first affecting the muscles of the hips, pelvic area, thighs
and shoulders. Calves are often enlarged.
 Progression - DMD eventually affects all voluntary
muscles, and the heart and breathing muscles.
Inheritance - X-linked recessive. DMD primarily affects
boys, who inherit the disease through their mothers.
Women can be carriers of DMD but usually exhibit no
symptoms.

16. Clinical Features
Genotype of DMD
 Females carry the DMD gene on the X
chromosome.
 Females are carriers and have a 50%
chance of transmitting the disease in
each pregnancy.
 Sons who inherit the mutation will
have the disease.
 Daughters that inherit the mutation
will be carriers.
 The DMD gene is located on the Xp 21
band of the X chromosome.
 Mutations which affect the DMD gene.
 30% are new mutations
 10-20% of new mutations occur in the
gametocyte (sex cell, will be passed
on to the next generation).
 The most common mutation are
repeats of the CAG nucleotides.

17. Genotype of DMD
(Cont.)
 During the translocation process, a mutation
occurs.
Mutations leading to the absence of dystrophin
 Very Large Deletions (lead to absence of dystrophin)
Mutations causing reading errors (causes a
degraded, low functioning DMD protein molecule)
 Stop mutation
 Splicing mutation
 Duplication
 Deletion
 Point Mutations

18. Clinical Features
Phenotype of DMD
 Delays in early childhood stages involving muscle use, in 42% of patients.
 Delays in standing alone
 Delays in sitting without aid
 Delays in walking (12 to 24 months)
 Toe walking or flat footednees.
 Child has a hard time climbing.
 Learning difficulties in 5% of patients.
 Speech problems in 3% of patients.
 Leg and calf pain.
 Mental development is impaired. IQ’s usually below 75 points.
 Memory problems
 Carrying out daily functions
 Increase in bone fractures due to the decrease in bone density.
 Increase in serum CK (creatine phosphokinase) levels up to 10 times
normal amounts.
 Wheelchair bound by 12 years of age.
 Cardiomyopathy at 14 to 18 years.
 Few patients live beyond 30 years of age.
 Reparatory problems and cardiomyopathy leading to congestive heart failure are
the usual cause of death.

19. Molecular Makeup
 There are 79 exons: which makeup 0.6% of the entire gene.
 There are 8 promoters (binding sites).
 Introns: make up 99.4% of the entire gene.
 Genomic DNA: 2.2 million base pairs.
 N-terminal or actin binding site: binds dystrophin to membranes surrounding
striated muscle fiber.
 Rod Domain: contains 24 proteins that repeat and maintain molecular
structure.
 It is thought to give the rod its flexibility.
 The main rod is interrupted by 4 hinge regions.
 The cysteine-rich domain: regulates ADAM protease which are cell
membrane anchors that are important in maintaining cell shape and
structure.
 The C-terminal: contains the syntrophin binding site (for binding internal
cellular components)

20. DMD Gene and Dystrophin
Function
 The DMD gene encodes for the protein
dystrophin, found in muscle cells and some
neurons.
 Dystrophin provides strength to muscle cells by
linking the internal cytoskeleton to the surface
membrane.
 Without this structural support, the cell membrane
becomes permeable. As components from outside
the cell are allowed to enter the internal pressure
of the cell increases until the cell bursts and dies.
 Under normal wear and tear stem cells within the
muscle regenerate new muscle cells and repair the
damage.
 In DMD the damage to muscle cells is so extreme
that the supply of stem cells are exhausted and
repair can no longer occur.

21. Jason’s family tree
Jason
15.11.99
Rachel
14.3.74
Tony
26.9.73
Susie
3.6.95
Fay
7.1.98
Jason, aged 3 yrs referred to a paediatrician by his GP
•Later walking than older siblings
•Difficulty running and riding tricycle
•Always climbs up stairs on all fours.

22. Fig. 1.4 ©Scion Publishing Ltd
Histology photos courtesy of Dr Richard Charlton.
Duchenne muscular dystrophy
(a) Affected boys stand up by bracing their arms against their legs (Gower’s manoeuvre) because their proximal
muscles are weak.
(b) and (c) Muscle histology (Gomori trichrome stain). Normal muscle (b) shows a regular architecture of cells
with dystrophin (brown stain) on all the outer membranes. (c) Shows muscle from a 10-year-old affected boy.
Note the disorganisation, invasion by fibrous tissue and complete absence of dystrophin.
Histology photos courtesy of Dr Richard Charlton, Newcastle upon Tyne.

23. Fig. 7.11 ©Scion Publishing Ltd
Photo. courtesy of Dr Richard Charlton
A muscle biopsy from a female carrier of Duchenne muscular dystrophy stained with
an antibody against dystrophin
Note the patchy distribution of staining around the outer membranes of cells (compare with the sections from
an affected boy and a normal control in next slide).

24. Immunolabeling of Muscle Biopsy Sections
Normal Control
Dystrophin
antibody staining of
muscle cells
4 year old boy with DMD – No detectable
dystrophin

25. Fig. 3.9 ©Scion Publishing Ltd
A deletion of part of the dystrophin gene
This figure shows a 500 kb region containing exons 41-50. These exons are all 100-200 bp long, and so if drawn to
scale each exon would be represented by a line occupying less than 0.05% of the width of the figure. Random
deletion breakpoints therefore almost always fall in introns. Their effect is to remove one or more complete
exons from the mature mRNA. The deletion shown removes exons 45-47 from the mature mRNA, while leaving
all the other exons intact.

26. Fig. 6.4 ©Scion Publishing Ltd
The dystrophin molecule anchors
the cytoskeleton of muscle cells
to the extracellular matrix, via the
dystrophin glycoprotein complex.
This includes the sarcoglycans
(mutations in which cause limb-
girdle muscular dystrophies) and
dystroglycans.
Muscle cells that lack dystrophin
are mechanically fragile, and fail
after a few years, hence
progressive muscle weakness.

27. The Beginning of Gene Therapy
for DMD
Advances in Gene Therapy
 Researches have developed "minigenes,"
which carry instructions for a slightly
smaller version of dystrophin, that can fit
inside a virus
 Researchers have also created the so-
called gutted virus, a virus that has had
its own genes removed so that it is
carrying only the dystrophin gene
Problems with Gene Therapy
 Muscle tissue is large and
relatively impenetrable
 Viruses might provoke the
immune system and cause the
destruction of muscle fibers with
the new genes

28. Fig | Summary of the wide
range of approaches being
used to treat Duchenne
muscular dystrophy. IGF1,
insulin-like growth factor 1;
NOS, nitric oxide synthase;
TNF-α, tumour necrosis
factor-α.

29. The real Deal in the new gene therapy
advances
In the past decade many trials have been made in the
muscular dystrophy area, particularly on DMD. The main
direction involves the Antisense technique. In a previous
presentation I introduced to you one mode of action of the
Antisense method which was EXON SKIPPING. So in the
remaining of this presentation we shall focus on the
researches and studies that investigated this technique

31. As mentioned earlier, DMD is caused by mutations in the dystrophin gene
that result in the absence or expression of mutant forms of the protein. The
human DMD locus at Xp21 spans approximately 2.5 million base pairs and
is the largest identied gene. It consists of 79 exons and the corresponding
14-kb transcript is expressed predominantly in smooth, cardiac and skeletal
muscle with lower levels in the brain.
Approximately 60% of DMD mutations are large insertions or deletions and
approximately 40% are point mutations or small frameshift rearrangements.
A commonality is that these mutations disrupt the translation reading frame
and lead to a truncated protein which is often not observed, possibly due to
protein instability or to nonsense-mediated mRNA decay. In contrast,
mutations, including large deletions, that do not disrupt the reading frame
and retain partly functional dystrophin expression usually lead to the milder
Becker muscular dystrophy (BMD) phenotype.

32. A sneak peek at the main
idea of exon skipping

34. THE NEXT PHOTO IS ELUSIVE, SO
BEWARE!
Now back to Dystophin

35. ERM family
Spectrin
family
e.dystrophin
muscular dystrophy

36. 3D Images of The Actin Binding
Sight Of Dystrophin

37. ANIMAL MODELS
 The most commonly studied animal models
of dystrophin mutations are the mdx mouse,
which carries a nonsense mutation in exon
23, and the Golden retriever muscular
dystrophy model (GRMD), which was found
to have a splice site mutation that leads to
exon 7 exclusion and a subsequent mRNA
frame-shift.PHOTO:
mdx mouse
PHOTO:
GRMD

39. (a)

41. Antisense derivatives of U7 and other small nuclear
RNAs as tools to modify pre-mRNA splicing patterns
In DMD-related mutations,
nonsense (as in mdx mice)or
frameshift mutations as well as
deletions (double wavy line) for
which the remaining exons are
out of frame can interrupt the
reading frame and cause a lack
of dystrophin. In many cases
the reading frame and hence the
production of an internally
truncated but partly functional
dystrophin can be restored by
skipping one or sometimes
multiple exons.

42. Trial for muscle mass increment
Adeno-associated virus can deliver the
gene forfollistatin and, because it is a
secretory peptide, very high serum levels
can be reached, resulting in muscle
hypertrophy at regional and remote sites.
When virus is injected into the
gastrocnemius and hamstring muscles in
animals, substantial increases in muscle
mass are noted at the site of injection and
at remote sites(Figure).We are exploring
AAV-mediated gene therapy as a form
of combinational treatment of muscular
dystrophy that would also be applicable
to other forms of muscle disease.
This immune response was due
to the circulating neutralizing
antibodies which forestall the
virion from binding to the
receptor on the target cell.
To reduce this effect, delivering
the virus through an intravascular
balloon catheter imparts a
protected environment enhanced
by the removal of blood from the
gene-targeted area, providing a
hospitable environment for
transduction
Follistatin is an antagonist of
Myostatin, which is an important
protein that limits the muscle
development. So when they injected
the model with rAAV1 the muscle
mass increased

43. Developments in Exon skipping
 When we talk about the
pioneers in the exon
skipping mechanism for
DMD we are talking
about the Dutch, who in
Leiden are making life
changing studies in this
aspect. A new antisense
oligonucleotide that is
being investigated for
phase III trials is
PRO051.
In October 2009, Prosensa partnered
with GSK for the development of its lead
compound PRO051. Both parties are
working closely together to make this
drug available to patients.
Prosensa’s lead compound
PRO051/GSK2402968 is highly
sequence-specific, i.e. no 100% full
length hits elsewhere in the human
genome, reducing the risk for off-target
effects. PRO051/GSK2402968 thus
specifically induces exon 51 skipping in
the DMD gene, which, given the
frequencies in various international DMD
mutation databases, could in principle
correct the reading frame in ~13% of all
DMD patients, including patients with
deletions of exon 50, exon 52, exons 45-
50, exons 48-50, and exons 49-50.
Clinical proof of concept
was obtained in four DMD
patients receiving a single
intramuscular 0.8 mg dose
of PRO051/GSK2402968
[van Deutekom et al., 2007].
In this study
PRO051/GSK2402968 was
safe, well-tolerated, and
effective in specifically
inducing exon 51 skipping
and dystrophin restoration
(up to 35% of normal) in the
majority of muscle fibers (up
to 94%) in the treated area.
Prosensa’s second product in
development, PRO044, also highly
sequence-specific, specifically induces
exon 44 skipping in the DMD gene. This
is applicable to 6% of all DMD patients,
including patients with deletions of exon
43, exon 45, exons 38-43, exons 40-43,
exons 42-43, and exons 45-54. Similar to
PRO051/GSK2402968, PRO044 is
extensively tested in vitro in series of
cultured muscle cells from patients with
different relevant mutations, and in the
hDMD mouse model.

44. PRO051 in depth
Figure. A new antisense
oligonucleotide,
PRO051, is promising
as a proof of principle
for jump-starting
production of the
dystrophin protein
lacking in the muscle of
patients with Duchenne
muscular dystrophy
(DMD
Figure. SCHEMATIC
REPRESENTATION OF
EXON SKIPPING.(Panel A)
In a patient with Duchenne
muscular dystrophy who has
a deletion of exon 50, an out
of-frame transcript is
generated in which exon 49
is spliced to exon 51. As a
result, a stop codon is
generated in exon 51, which
prematurely aborts
dystrophin synthesis.(Panel
B) The sequence-specific
binding of the exon-internal
antisense oligonucleotide
PRO051 interferes with the
correct inclusion of exon 51
during splicing so that the
exon is actually skipped.This
restores the open reading
frame of the transcript and
allows the synthesis of a
dystrophin similar to that in
patients with the clinically
milder Becker muscular
dystrophy.
Figure. MECHANISM OF PRO051 IN THE
RESTORATION OF DYSTROPHIN
EXPRESSION THROUGH EXON
SKIPPING.(Panel A) Normal muscle produces
dystrophin in response to signals encoded by the
enormous DMD gene, in which 79 exons are
spliced together in messenger RNA (mRNA).
The mRNA is then translated into dystrophin
protein. All exons are spliced to maintain the
triplet codon reading frame required for effective
protein translation.(Panel B) In the muscle of
patients with DMD, mutations in the dystrophin
gene lead to the loss of one or more exons. The
mRNA splices together the remaining exons;
however, the triplet reading frame in not
maintained, which leads to errors in translation
(frame shift) and loss of production of the
dystrophin protein. The loss of dystrophin at the
plasma membrane leads to the secondary loss of
other associated membrane cytoskeleton
structures. This, in turn, leads to membrane
fragility, an abnormal influx of calcium ions and
the efflux of creatine kinase into the patient's
blood.(Panel C) Intramuscular injection of
PRO052 probably enters the Duchenne muscle
through abnormal muscle membranes; it then
enters the nucleus and binds to the dystrophin
mRNA in a sequence-specific manner. PRO051
blocks the splicing machinery, preventing the
inclusion of an additional exon (exon 51 in this
example). The skipping of this additional exon
restores the reading frame of the mRNA,
allowing new production of dystrophin.
Orphan Drug Designation
Was Given to PRO051;
meaning that it is a possible
cure for one of the rare
diseases

45. a | In Duchenne muscular
dystrophy (DMD) patients with
a deletion of exons 45–54, an
out-of-frame transcript is
generated in which exon 44 is
spliced to exon 55. Owing to the
frame shift, a stop codon occurs
in exon 55, which prematurely
aborts dystrophin synthesis. b |
Using an exon-internal antisense
oligonucleotide (AON) in exon
44, the skipping of this exon can
be induced in cultured muscle
cells. Accordingly, the transcript
is back in-frame and a Becker
muscular dystrophy (BMD)-like
dystrophin can be synthesized
b,c | Exon skipping also led to substantial dystrophin synthesis in situ,
which was detected by the immunohistochemical analysis of treated
myotubes (b) and by Western blot analysis of protein samples from
treated myotubes (c) using the Dys2 antibody (raised against the distal
exons 77–79). The dystrophin was located at the membrane at two days
(2d) and accumulated up to seven days (7d) post-transfection. No
dystrophin was observed in untreated samples (NT). As expected from the
deletion, the dystrophin that was produced was shorter than the full-length
dystrophin from a (1:10 diluted) human control sample (HC).

46. Other antisense oliigonucleotides being
developed for dmd
AVI BioPharma is developing AVI-4658
for the treatment of DMD. This first
generation PMO drug candidate is
designed to skip exon 51 of the
dystrophin gene, allowing for restoration
of the reading frame in the dystrophin
mRNA sequence. Results from a Phase
1 proof-of-concept trial showed that
injection of the drug into the muscles of
a series of boys with DMD successfully
induced dystrophin production in a
dose-responsive manner. Further, the
drug was well tolerated, with no
significant drug–related adverse events
detected. AVI is currently conducting an
ongoing Phase 1b/2 dose-finding
clinical trial evaluating the systemic
delivery of AVI-4658 for treatment of
DMD.
AVI BioPharma is also
developing a second
generation chemistry exon
skipping drugs, with a PPMO,
AVI-5038, nearing IND
submission for the treatment of
DMD by skipping exon 50
Three patients, one each in the 2.0, 10 and 20 mg/kg cohorts,
demonstrated substantial generation of new dystrophin-positive
muscle fibers, including the first ever reported generation of
dystrophin-positive muscle fibers of more than 50% of normal in a
patient following systemic administration of a drug.
All 8 patients in the 10 and 20 mg/kg cohorts demonstrated
generation of new dystrophin-positive muscle fibers.
The three patients, one each in the 2.0, 10 and 20 mg/kg cohorts,
demonstrating substantial generation of new dystrophin-positive
muscle fibers had multiple fold increases in dystrophin protein
expression measured by Western blot over baseline, with patients
in the 20 mg/kg cohort demonstrating the highest increases. These
three patients also had noted increases in dystrophin per fiber.

48. PATIENT BEFORE
TREATMENT
PATIENT AFTER TREATMENT