Current muscles and their protection from injury during

Current therapeutic
strategies for Duchenne Muscular Dystrophy


Duchenne Muscular
Dystrophy (DMD) is an X-linked, genetically inherited, debilitating, life
shortening disease caused by a malfunction of the DMD gene. The DMD gene is the
longest single gene in the human body which codes for dystrophin: a rod-shaped
cytoplasmic protein, the protein is usually involved in joining actin fibres from
the cytoskeleton and intracellular matrix contractile apparatus to the
extracellular matrix1. Histological signs can be identified of DMD
prior to any pathophysiological processes of the disease. In fact, foetal
diagnosis can be carried out as early as the third trimester of pregnancy. There
are many exciting methods of potential treatment for DMD, the two most promising
at present could are those that involve the use of viral vectors or modified
nucleic acids.


Pathophysiology of DMD

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Dystrophin is part of an assembly of proteins responsible
for strengthening cardiac and skeletal muscles and their protection from injury
during muscular contraction and relaxation. Specifically, dystrophin acts as an
anchor protein, as stated and also can be found in nerve cells in the brain
however their function is not affirmed however research suggests that they may
be associated with the usual function of synaptic transmission2.

When Dystrophin is solubilised from its sarcolemmal fraction it becomes associated
with glycoproteins and a large oligomeric complex, this forms the dystrophin
associated glycoprotein complex. This, in essence, forms the critical link
between the cytoskeleton and intracellular matrix contractile apparatus to the
extracellular matrix in order for muscles to carry out persistent, undamaging
muscular contraction.

Therefore, when the dystrophin associated glycoprotein complex
malfunctions, due to a dystrophin problem, the intuitive symptomatic features
would be muscular related. The cause of the dystrophin protein being
non-functional or partially functioning is due to a gene mutation in the DMD
gene, most commonly a deletion, followed by point mutation and duplication
respectively. DMD is characterised by, most commonly, an abnormal gait,
frequent falls and difficulty climbing steps. Less common symptomatic findings
include a reluctance to walk, poliomyelitis, delayed walking, walking on toes
and excessive fatigue. Lumbar Lordosis is commonly affiliated with the waddling
gate found in DMD patients. Patients are often able to sustain a short period
of time stood on one leg however are unable to hop or turn in a usual form. The
most illustrative observational sign of DMD may be ‘the Gowers’ Manoeuvre’
which shows a stereotypical sequence of postures shown in rising from a prone
position to an erect posture. However, Gower’s Manoeuvre is not pathognomonic
as it is also present in other muscular disorders. The six-minute walk test (6MWT)1,
a recording of the distance that a patient can walk within six minutes, can be
used as a method to monitor the progression of a disease such as DMD. DMD
affects around 1/5000 male births and those affected rarely live into their
fourth decade of life. Thus, there is clearly a desire for an effective
treatment to halt this drastically life shortening life.


Methodologies for muscular gene delivery

There are a variety
of methods that involve the delivery of genes, specifically the DMD gene into
the human muscular system. One of the most exciting methods of gene insertion
is using an Adeno Associated Virus (AAV), an asymptomatic, small virus which
can be modified by inserting DNA to be used as a vector to deliver sections of
DNA into the human body. It is widely used due to its overall efficacy and
safety. The issue with DMD and using an AAV to deliver the DMD gene is the
relative size of both subjects. The DMD gene is a very large gene: 2.3
megabases, the full-length cDNA is over 11kilobases whereas the AAV virus can
usually capacitate just less than 5 kilobases. The question arises therefore
how do we effectively deliver the DMD gene, via AAV in attempt to treat DMD.

One method, seems to be to divide the full-length cDNA into three segments,
each inserted into a vector (AAV) which all have unique recombination signals
for directional recombination: Triple transplicing and reconstitution. This
method benefits due to the entire reconstitution of the full-length dystrophin
being successfully expressed in mice. However, the efficiency of this method
must be questioned due to low rates of successful vectoral reconstitution,
however over time stronger and more unique recombination signals may lead to
greater effectiveness rates. Equally as exciting is the report that there is
the potential that AAV can be engineered to express up to 15kb of gene, three
times greater than that of a wild-type AAV genome, therefore further
optimisation of these methodologies may lead to expansion of the use of AAV in
gene therapy3. The question asks however, is there a potential viral
vector which either has a greater capacity for carrying DNA or is there a viral
vector which responds greater to unique recombination signals, the latter I
feel has been less investigated and therefore may lead to a potential new
pathway of research.


An alternative
approach has also been made, using AAVs, to deliver a treatment of DMD. This
method involves using micro and mini-dystrophin, within an AAV vector in an
attempt to address the symptoms of DMD. Using mini-dystrophin is aimed at the
reduction of the symptomatic effects of DMD to relate them more to those
experienced in mildly affected Becker’s Muscular Dystrophy (BMD). Micro-dystrophin,
however, aims at the minimum requirement of the gene to function as normal
dystrophin and that even in 50% correction would lead to a sufficient treatment
of cardiomyopathy in mdx mice4.

In summary AAV can be used as a mediator to combine separate parts of the DMD
gene or to carry truncated versions of the DMD gene in attempt to treat DMD
however not eradicate the condition.


oligonucleotides (AONs), are nucleic acids, single stranded and chemically
modified to target specific gene transcripts. Its small size is essential for
delivery and the chemical modifications that occur can affect: toxicity,
affinity, solubility, stability and degradation resistance1. There
seem to be two main leading AONs: 2′-O-methyl phosphorothioate (2OMePs) and
phosphorodiamidate morpholino oligomers (PMOs). The main difference between the
two seems to be 2OMeP’s negative charge which leads to interactions with many
proteins, in particular the modulation of TLRs and leading to inflammatory
symptoms, renal failure and thrombocytopenia5. Therefore, it seems strange
to consider 2OMePs when PMOs induce little side effects whilst treatment showed
positive signs of revertant fibres in patient studies6. Therefore,
there must be further research into which of PMOs and 2OMePs are more efficient
or whether a cocktail of both AONs may be effective and if so whether there is
a method of preventing the modulation of TLRs by 2OMePs.




Relative Pre-Clinical Evidence


In terms of AAV,
beyond using mice with DMD, dogs become the next subject prior to clinical
trials due to their greater complexity. In terms of evidence the aim is to
evaluate which serotype of AAV does not initiate a strong immune response,
leading to a selectin of an AAV which will not be destroyed and potentially
that will remain to promote Dystrophin creation for a longer period. AAV-1
showed no protein expression when administered as a local limb muscle injection
and limb perfusion, in a young adult dog7. AAV-2 was carried out on normal
dog muscle and led to an expression of cytotoxic T lymphocytes8.

Most AAVs seem to either have a strong CTL production with no protein
expression. However, AAV-8 had no CTL production and expression occurred
however only for 2 minutes and was only on one specimen9. Therefore,
it seems clear that to progress, more investigation needs to be taken on
serotypes of AAV possibly specifically fine-tuning serotype AAV-8, as well as
their ability to carry micro- and mini-dystrophin and their potential ability
to function via triple or potentially double trans-splicing.


In terms of using
AONs in pre-clinical research there is much compelling positive evidence. For example,
in mdx52 mice using an injection
containing the 10 vPMO cocktail led to a 55% increase in the number of
dystrophin positive fibres produced. Moreover, Western Blotting shows that
using 10 vPMO can lead to full-length dystrophin expression10. Moreover,
evidence that no adverse side effects were present in ten-fold doses in
non-human primates indicates the safety of AONs and their suitability for use
in fully clinical trials. Primates were euthanized in extremis using ketamine and an IV overdose of sodium
pentobartbital solution. The skin of the primates was then reflected from a
ventral midline incision and any abnormalities were identified. 59 different
tissue samples were examined. No effects related to treatment with AVI-4658
were observed with respect to clinical observations11. This clearly
shows that the next step from these observations would be to carry out a human
clinical trial to monitor the efficacy, potency and the ability to remain in
the body by AONs and, seemingly more specifically, PMOs.


Clinical Evidence

There are currently
many clinical trials in action and in different stages aiming at treating DMD,
including trials concentrating on the AAV and AON methodology mentioned in this
essay. However due to DMD research being a pioneering medical process many
trials have not had their results published or are in the further stages of
clinical trials. Using PMOs, specifically Eteplirsen, exhibited some
improvement in 7 of 19 patients. On average dystrophin fluorescence doubled in
patients on 2mg/kg/week and patients treated with 30 mg/kg/week had a
significant 22.9% increase 24 weeks posttreatment. There was no such increase
in the placebo grouping. After 48 weeks, there was a 51.7% increase. There was
no additional benefit for patients treated with 50mg/kg/week. However due to
some patients losing ambulation it was not a satisfactory end point of the
train as determined by the FDA. The main issue with PMOs seems to be the challenge
presented by increased tissue-target uptake, this is due to the rapid clearance
of PMOs due to their neutral nature12.


In terms of AAV, there are various research programmes
currently in session, mainly by Dr Jerry Mendell, including the administration
of rAAVrh74.MCK,µDystrophin13 intramuscularly to
investigate whether this viral vector could be used as a potential treatment
for DMD. Moreover, the trial of rAAV1.CMV.huFollistatin34414
will be used by Dr Mendell. Once these trials have been recorded, reviewed and
published we may gain a greater insight on how far we have to go towards an
effective treatment of DMD.




There are many
different viewpoints to be looked at when investigating the potential for a
cure/treatment of DMD. Challenges are presented due to the infancy of the
techniques used and also due to the systemic nature of the disease. There are,
of course, other genetic methods that potentially may be able to treat DMD,
such as CRISPR-Cas9, however it seems that the use of AONs, specifically PMOs
in addition to the promising use of AAVs and their potential manipulation are
the most fore thinking and possibly closest to a true progression that may
result in a beneficial alteration of the prognosis of DMD.





Robinson-Hamm J, Gersbach C. Gene therapies that restore dystrophin expression
for the treatment of Duchenne muscular dystrophy. Human Genetics.


2. Reference G.

DMD gene Internet. Genetics Home Reference. 2017 cited 12 November 2017.

Available from:

3. Lostal W,
Kodippili K, Yue Y, Duan D. Full-Length Dystrophin Reconstitution with
Adeno-Associated Viral Vectors. Human Gene Therapy. 2014;25(6):552-562.


Athanasopoulos T, Graham I, Foster H, Dickson G. Recombinant adeno-associated
viral (rAAV) vectors as therapeutic tools for Duchenne muscular dystrophy
(DMD). Gene Therapy. 2004;11(S1):S109-S121.

5. Kole R,
Krieg A. Exon skipping therapy for Duchenne muscular dystrophy. Advanced Drug
Delivery Reviews. 2015;87:104-107.

6. Fall A,
Johnsen R, Honeyman K, Iversen P, Fletcher S, Wilton S. Induction of revertant
fibres in the mdx mouse using antisense oligonucleotides. Genetic Vaccines and
Therapy. 2006;4(1):3.

7. Vulin A,
Barthélémy I, Goyenvalle A, Thibaud J, Beley C, Griffith G et al. Muscle
Function Recovery in Golden Retriever Muscular Dystrophy After AAV1-U7 Exon
Skipping. Molecular Therapy. 2012;20(11):2120-2133.

8. Yuasa K,
Yoshimura M, Urasawa N, Ohshima S, Howell J, Nakamura A et al. Injection of a
recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune
responses against transgene products. Gene Therapy. 2007;14(17):1249-1260.

9. Koo T, Okada
T, Athanasopoulos T, Foster H, Takeda S, Dickson G. Long-term functional
adeno-associated virus-microdystrophin expression in the dystrophic CXMDj dog.

The Journal of Gene Medicine. 2011;13(9):497-506.

10. Aoki Y,
Yokota T, Nagata T, Nakamura A, Tanihata J, Saito T et al. Bodywide skipping of
exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery.

Proceedings of the National Academy of Sciences. 2012;109(34):13763-13768.

11. Sazani P,
Ness K, Weller D, Poage D, Palyada K, Shrewsbury S. Repeat-Dose Toxicology
Evaluation in Cynomolgus Monkeys of AVI-4658, a Phosphorodiamidate Morpholino
Oligomer (PMO) Drug for the Treatment of Duchenne Muscular Dystrophy.

International Journal of Toxicology. 2011;30(3):313-321.

12. Lim K,
Maruyama R, Yokota T. Eteplirsen in the treatment of Duchenne muscular
dystrophy. Drug Design, Development and Therapy. 2017;Volume11:533-545.

13. Clinical
Intramuscular Gene Transfer Trial of rAAVrh74.MCK.Micro-Dystrophin to Patients
With Duchenne Muscular Dystrophy – Full Text View –
Internet. 2017 cited 12 November 2017. Available from:

14. Clinical
Intramuscular Gene Transfer of rAAV1.CMV.huFollistatin344 Trial to Patients
With Duchenne Muscular Dystrophy – Full Text View –
Internet. 2017 cited 12 November 2017. Available from: