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\begin{document}

\title{The effect of emerging therapies on cardiopulmonary disease in Duchenne
muscular dystrophy}



\author[1]{Doug McKim}%
\author[2]{Timothy Cripe}%
\author[3]{Linda Cripe}%
\affil[1]{Ottawa Hospital Research Institute}%
\affil[2]{Nationwide Children's Hospital}%
\affil[3]{Nationwide Children’s Hospital}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Gene therapy is an attractive approach being intensively studied to
prevent muscle deterioration in patients with Duchenne muscular
dystrophy. While clinical trials are only in early stages, initial
reports are promising for its effects on ambulation. Cardiopulmonary
failure, however, is the most common cause of mortality in DMD patients,
and little is known regarding the prospects for gene therapy on
alleviating DMD-associated cardiomyopathy and respiratory failure. Here
we review current knowledge regarding effects of gene therapy on DMD
cardiomyopathy and discuss respiratory endpoints that should be
considered as outcome measures in future clinical trials.%
\end{abstract}%



\sloppy




\textbf{Introduction}

Duchenne muscular dystrophy (DMD) is a rare X-linked genetic disorder
that results from out of frame mutations in the dystrophin gene. The
incidence is believed to be 1 in 5,000 male births \textsuperscript{1};
however, it is now appreciated that female carriers can also manifest
the disease. Dystrophin is a critical protein involved in membrane
stabilization by anchoring the inner surface of the sarcolemma to
F-actin \textsuperscript{2}. Disruption of the protein results in both
progressive skeletal muscle disease as well as a cardiomyopathy. Modern
advancements in respiratory therapies have significantly impacted
survival with the cardiomyopathy now considered the leading cause of
mortality. While the focus historically has been largely on the impact
of therapies on skeletal muscle disease, with the advent of emerging
molecular and genetic therapies, clinical trials will need to focus on
understanding the impact not only on skeletal muscle function but also
on cardiopulmonary disease. The failure of many trials to include
cardiac and even sometimes respiratory endpoints has impaired our
ability to understand the impact of contemporary therapies on the heart
and lungs.

If there is not a family history of the disease, children tend to be
diagnosed within the first few years of life when they fail to meet
gross motor milestones. Clinical cardiac dysfunction tends not to occur
later in the disease, but we now know that the myocardium is impacted
early in the disease. In fact, EGC's taken in the newborn and young
child are often abnormal and show evidence of left ventricular
hypertrophy. Furthermore, cardiac magnetic resonance imaging (CMR) has
allowed us an important window into what is occurring in the heart of
the young DMD patient. Abnormalities of myocardial strain are noted
before the onset of the development of myocardial fibrosis or declining
function. Traditional heart failure medications are the only avenue of
treatment at this time. Left ventricular assist devices (LVAD) and
cardiac transplantation are treatments that have been utilized in
isolated instances.

Early reports of new advances in gene therapy appear promising for
delaying loss of ambulation and improving quality of life in children
with DMD \textsuperscript{3}. The impact of gene therapy on
cardiopulmonary outcomes, howver, is unknown. Herein we review
preclinical data regarding the potential for gene therapy to ameliorate
DMD-associated cardiomyopathy and review the landscape of current DMD
gene therapy efforts, including gene replacement, gene modulation, and
gene editing. Because even less is known about the effects of gene
therapy on lung function, we focus our discussion on various pulmonary
endpoints that should be considered as potential outcome measures in
future trials of DMD gene therapy.

\textbf{Gene Therapy and the DMD Heart}

The promise of gene therapy is to restore expression of a normal protein
in a cell containing a genetically deficient or defective version. In
that sense, gene therapy raises the possibility of a cure for DMD, not
only for skeletal muscle but also for the heart. Strictly speaking, gene
therapy is use of nucleic acid, either DNA or RNA (or both) to alter the
function of a cell. A variety of approaches have been tried to carry
nucleic acid into a cell, including direct injection of DNA (so-called
``naked'' DNA), DNA transfected into a cell by compacting it with a
glycoprotein/polycation scaffolding, encapsulated in liposomes that fuse
with the cell membrane, or most commonly packed in a virus shell.
Viruses have evolved to efficiently enter cells and transmit DNA to the
nucleus, and many can be ``gutted'' of many or all of their normal genes
to make room for transferring in new genes. The new gene is referred to
as a ``transgene,'' virus-mediated entry into a cell is termed
``transduction,'' and the virus carrying the transgene is called a
``vector.'' While many different viruses have been used for
transduction, a small member of the parvovirus family, adeno-associated
virus (AAV), is most commonly used due to its safety profile (it is not
associated with any disease), efficiency of transduction, widespread
biodistribution following intravenous delivery, and ease of
manipulation. Whereas the normal ``wild type'' adeno-associated virus
tends to integrate its DNA into a specific locus on chromosome 19,
gutted versions containing a transgene persist in the nucleus as
circular episomes, separate from the cellular chromosome. This lack of
integration markedly reduces the ``genotoxicity,'' meaning the chance
for causing genetic mutations in cellular genes, but it also increases
the chance the new gene could be ``lost,'' since it is not permanently
incorporated into the cellular genome.

Critical to the success of gene therapy is ensuring adequate expression
in the diseased cell, meaning skeletal and heart muscle cells in
patients with DMD. There are two major determinants of adequate
expression, the AAV serotype and promoter. The serotype (a word
originally used to define related viruses distinct enough not to show
cross reactivity with immune serum) determines the amino acid sequence
of the outer shell of the virus, which contains the ``key'' to enter a
cell; different serotypes bind to different cell surface receptors to
gain entry. While most but not all naturally occurring AAV serotypes
enter skeletal muscle cells, only a subset also enter heart muscle
cells. Each serotype has its own unique biodistribution, as do many new
synthetic serotypes being created and explored to preferentially
transduce specific tissues. A seemingly infinite variety of capsid
variations are possible, and finding those best suited for DMD is
underway \textsuperscript{4}. No virus has yet been discovered or
created that is completely specific for a given cell type, and most
viruses transduce liver cells after given intravenously due to features
of circulation and liver uptake, in addition to other tissues. Once the
virus binds and enters a cell, the second determinant regarding
expression is the activity of the enhancer/promoter in that cell. There
are many well-known naturally occurring strong enhancer/promoters, such
as from a cytomegalovirus gene, the elongation factor 1a gene, and
chicken \selectlanguage{greek}β-\selectlanguage{english}actin, as well as many synthetic and hybrid versions, that are
capable of activating gene expression across a wide variety of cell
types. While these promoter/enhancers might work well in muscle, they
may also express the transgene, in this case a small form of dystrophin,
in cells that don't normally express it, which could cause harm. Thus,
gene therapy vectors for DMD use enhancers/promoters derived from
muscle-specific genes such as muscle creatine kinase, desmin, and myosin
heavy chain, in order to restrict expression to muscle cells. Taken
together, in order for gene therapy to work in the DMD heart, it must be
comprised of a vector serotype that transduces cardiac muscle cells and
an enhancer/promoter that is active or ``on'' in those cells.

The choice and structure of the transgene is also an important
consideration, especially in DMD. Because the full-length dystrophin
protein is 3,685 amino acids, it requires 11,595 DNA nucleotide bases to
encode it (3 bases per amino acid). AAV shells are quite small, about 70
nanometers, and they can only package approximately 4,700 bases of DNA.
From this vantage point, some viruses with large packaging capacities
such as herpes simplex virus might seem more attractive, but many such
larger virus types do not transduce muscle cells well. The main solution
employed so far to solve this size dilemma has been to use a shortened
version of dystrophin. Much of the dystrophin protein within the
so-called rod domain is comprised of numerous repeats, which can be
deleted to fit into AAV. While these so-called mini-dystrophin (akin to
dystrophin in Becker patients) or micro-dystrophin (with nearly all
repeats deleted) proteins are not normal, they are sufficient to restore
significant function to muscle cells. Such an approach markedly improved
cardiomyopathy in aged mdx mice, resulting in improvements on
electrocardiography, hemodynamic measurements, and prevented
dobutamine-stress induced cardiac death \textsuperscript{5}.

Ongoing clinical development of gene therapy for DMD is currently being
spear-headed by three main companies. Sarepta is using an AAV serotype
derived from rhesus monkeys called rh74. An advantage of this serotype
due to it being a monkey virus is that, ostensibly, fewer people have
pre-existing immunity to AAVrh74 compared to human-derived AAV
serotypes. Their micro-dystrophin gene is driven by an engineered hybrid
promoter, MHCK7, derived by combining sequences from the creatine kinase
and alpha-myosin heavy chain complex promoters and shown to have high
expression in both cardiac and skeletal muscle cells\textsuperscript{6}.
Thus, it is anticipated this virus will be effective in treating the
heart. Similarly, Solid Biosciences uses an AAV9 serotype which enters
skeletal and cardiac cells as well as a variety of other tissues, and a
muscle creatine kinase 8 promoter, which also shows robust cardiac
expression in preclinical studies. In contrast, while the serotype being
used by Pfizer is also an AAV9 serotype, their mini-dystrophin gene is
driven by the human creatine kinase promoter that for which some data
suggest it is not particularly active in cardiac cells. This construct
may do well in skeletal muscle, but whether it significantly ameliorates
DMD-associated cardiomyopathy is unclear.

As an alternative to replacing the defective dystrophin gene with a
shortened version, another approach is to express a gene to help the
disease muscle cell in other ways. The GALGT2 gene encodes
glycosyltransferase that increases expression of a variety of proteins
that can improve muscle cell function such as utrophin, plectin1, agrin,
several laminins and integrin. Indeed, AAV-mediated gene transfer of
GALGT2 improves not only skeletal muscle pathology in the mdx mouse but
also is very effective at improving cardiac function
\textsuperscript{7}and is currently in clinical trials
(https://clinicaltrials.gov/ct2/show/NCT03333590).

Some dystrophin mutations are amenable to exon skipping; that is, their
alterations or deletions are contained within a single exon that
essentially derails the rest of the protein by changing the reading
frame and resulting in a premature stop codon. In such cases, antisense
oligonucleotides designed to specifically interfere with splicing of
that particular exon can cause it to be excluded from the final mRNA
during splicing, restoring the reading frame and thus the remainder of
the protein. In fact, a chemically stabilized antisense oligonucleotide
(so-called ``morpholino'') specific for exon 51 is FDA approved for
patients with mutations in that exon, and other antisense molecules are
being developed for other exons. The major limitation of this approach
is achieving sufficient quantities to enough muscle nuclei to have a
broad and high impact on restoring dystrophin expression, though
conjugation of the antisense with peptides may improve delivery. Another
approach to overcome this limitation is to place the antisense sequence
in a U7 small nuclear RNA expressed in an AAV, again leveraging the
virus biology to achieve high levels in the muscle nucleus.
Interestingly, the combination of both the peptide-conjugated morpholino
together with AAV-U7 appears even better than either approach
alone\textsuperscript{8}.

Due to recent biotechnological advances, over the past few years the
definition of gene therapy has expanded from one that is primarily about
``therapy using nuclei acids or genes,'' such as AAV-insertion of a
functional gene into the nucleus or anti-sense oligonucleotides to skip
an exon as described above, to one that encompasses ``therapy OF
genes.'' By leveraging and adopting a bacterial system of proteins and
template or ``guide'' RNAs, we can now precisely edit genes \emph{in
situ} ; that is, we can make deletions or insertions into the cellular
genome at specific locations based on the unique DNA sequence. Again a
nuclei acid is used as a therapeutic, as the hybridization to DNA, or
the ``matching'' of the guide RNA sequence to the DNA, determines where
in the DNA to cut and/or insert. Critical to the process is the
bacterial protein as well, known as the clustered regularly interspaced
short palindromic repeats (CRISPR)-associated protein (Cas). There are
several Cas family members, with Cas9 the most commonly one in use. This
new capability is rapidly transforming our vision of what diseases might
someday be treatable with gene therapy, including DMD. In fact,
encouraging proof-of-principle studies in mice have been published. For
example, in the mdx mouse that contains a mutation in exon 23 of
dystrophin, CRISPR/Cas9 was successful used to delete the mutant exon,
restore the reading frame, and partially restore dystrophin expression,
resulting in improved function of both skeletal and cardiac
muscle\textsuperscript{9,10}. While promising, there remain a number of
barriers to clinical use of CRISPR/Cas9 technology. The follow-up study
of those mice showed persistence, long-term expression one year later,
but detailed analysis also reviewed immunity to the bacterial Cas9
protein and unintended ``off target'' gene editing \textsuperscript{11}.
The latter has been a concern in the field since its inception, and
exactly how precisely we can edit the genome without inducing other
mutations that either could be damaging to the cell or cause other
diseases such as cancer is unknown. Another potential barrier to
CRISPR/Cas9 use in humans was recently uncovered: pre-existing immunity
to bacterial Cas9, given humans are colonized with a microbiome, which
in a mouse model destroyed cells edited with CRISPR/Cas9 due to
anti-Cas9 immunity \textsuperscript{12}.

\textbf{Respiratory Outcome Measures for Trials of Emerging Therapies in
Duchenne Muscular Dystrophy}

While the goal of gene modifying and molecular therapies for DMD is to
improve survival, the duration of prospective clinical trials are too
limited to be capable of demonstrating an impact on this outcome. As
such, surrogate measures of respiratory function which correlate with
survival and quality of life (QoL) are necessary. Measures must also be
sufficiently sensitive to change within the population for whom the
therapeutic effects can be the most meaningful, those younger
individuals with DMD.

In order to design clinical trials of novel therapies and assess their
impacts on respiratory function, it is necessary to appreciate the
natural history of the time course and evolution of respiratory muscle
and pulmonary function in DMD as well as the impact that standard
therapies may have on that evolution. Furthermore, and perhaps more
importantly, multiple individuals with identical genetic features may
have significantly different trajectories for cardiopulmonary function.
As such, it will be necessary to stratify randomization by phenotypic
behavior in order to more accurately evaluate the effect of emerging
therapies.

\textbf{Natural history of respiratory function in DMD}

A major measure of lung function is absolute vital capacity (VC), which
rises from age 7 and usually peaks between 12 and 14 years and then
declines due to progressive muscle weakness. While absolute VC continues
to rise, the percentage of ideal or predicted VC (VC\%pred) declines in
a linear fashion reaching about 80\% predicted at age
10\textsuperscript{13}. Hence, gradually falling VC\%pred values include
absolute values which occur before, at and following peak VC. Some
investigators suggest that the identification of absolute values before,
at and following peak VC may be best to assess the effect of
interventions \textsuperscript{14}. In a large cohort study the VC of
untreated individuals peaked at 1.85 liters at age 12 to 13 yrs and
began to decline when VC\%pred fell to 60-70\% \textsuperscript{15}.
Rates of decline of VC\%pred per year have varied in the literature
between as little as 5\% (ages 5 to 24 yrs) \textsuperscript{16} to as
much as 10.7\% (ages 12 to 15 yrs) \textsuperscript{17}. Following peak
VC, absolute decline in ml/yr varies between 118 \textsuperscript{14}and
180 mls/yr \textsuperscript{18} with subsequently both FVC and VC\%pred
reaching an asymptotic floor after 20 years of
age\textsuperscript{14-16,19,20}. Without ventilatory support, a more
favorable prognosis is associated with a higher, later peak and a more
gradual decline in VC \textsuperscript{21}. Consensus guidelines
recommend lung volume recruitment strategies at VC 60\%pred,
mechanically assisted coughing at 50\%pred and NIV below
50\%pred\textsuperscript{22}. A VC of less than 1 liter has been
associated with a 5 year survival of only 8\% without respiratory
supports\textsuperscript{18}. At VCs below a liter (680cc) up to 24 hour
respiratory support, preferably noninvasively, may be required. As such,
disease modifying therapies have the goal of increasing and delaying the
peak VC and reducing the post-plateau rate of decline. The more or less
linear decline of VC\%pred as an outcome measure may not be as sensitive
to change as a result of interventions, as absolute
VC\textsuperscript{13}.

Phenotypic differences in pulmonary function and therefore clinical
outcomes have been recognized for years. Phillips identified a longer
surviving population who experienced peak VC at a median of 2 years
later, 13 vs. 15 years and a slower yearly decline in VC\%pred, 6\% vs,
7.8\% \textsuperscript{18} and other investigators have divided DMD
populations in to cohorts with different outcomes and survival related
to the number of mutations upstream to exon 30 \textsuperscript{23}.
Even within the same genotype heterogeneity exists and needs to be
considered in the evaluation of emerging therapies \textsuperscript{23}.

\textbf{Effect of standard therapies on pulmonary functions and clinical
outcomes}

Glucocorticoid (GC) therapy is well established to help delay and
increase the maximum VC plateau though this is then followed by the same
rate of loss. McDonald found that compared to untreated individuals
whose VC peaked at 1.85 liters between ages of 12.0 to 12.9 yrs, those
treated with GCs had a peak median FVC of 2.03 liters at age 17.0 to
17.9 years, both increasing and delaying the maximum VC. As well, the
age at which VC fell below 1 liter was delayed from 20.0-20.9 until
23.0-23.9 years \textsuperscript{16}.

In addition to regular monitoring of pulmonary function and gas
exchange, international guidelines recommend preventive and supportive
respiratory therapies for individuals with DMD; lung volume recruitment
strategies, manual and mechanical cough assistance and noninvasive
ventilation (NIV) \textsuperscript{22,24}. A number of these therapies
have effects on respiratory function and its evolution.

\textbf{Lung Volume Recruitment (LVR).} LVR, the active or passive
stacking of breaths with a resuscitation bag or mouthpiece ventilator in
order to achieve a maximum insufflation capacity (MIC) has been
demonstrated to have a beneficial effect, not only on cough flows but on
lung mechanics. There is evidence for short term improvement in
respiratory system compliance and observational evidence for a
longer-term effect on important measures such as rate of loss of VC as a
result of LVR.

Molgat-Seon demonstrated that respiratory system compliance can be
significantly improved for an hour after a session of
LVR\textsuperscript{25} and Chiou showed that the peak/plateau VC can be
delayed by 5 years and the post-peak rate of decline significantly
decreased \textsuperscript{15}. The mechanism for improvement in loss of
VC is most likely related to the establishment and maintenance of a
maximum insufflation capacity (MIC). The MIC-VC difference and the
resulting peak cough flow are the measures of effectiveness of LVR. In a
small retrospective study, McKim demonstrated that the rate of decline
of VC \%pred after age 18 can be reduced to 0.5\% per
year\textsuperscript{26} compared to a recent cohort of 47 DMD subjects
not using GCs with mean age of 17.5 \selectlanguage{ngerman}±4.7 years and a mean rate of FVC\%p
decline of 4.68\%/yr \textsuperscript{27}. In addition, the MIC-VC
difference can be maintained for over 8 years at a level sufficient to
achieve effective cough flows \textsuperscript{28}.

\textbf{Noninvasive ventilation (NIV).} NIV has provided the most
profound effect on survival in DMD, from the late teens to at least the
late 20's, even into the 40s in the presence of favorable cardiac
function and 24 hour, continuous NIV/mouthpiece ventilation, in spite of
little or no measurable pulmonary function \textsuperscript{29-31}.
Initiation of NIV can be considered, in pharmacotherapy trials, as an
important outcome to be delayed as a consequence of improved muscle
function. However, if necessary and initiated, NIV can have important
and significant effects on sleep quality, quality of life and, of course
on survival, even with no measurable VC \textsuperscript{32,33}.

A small number of publications indicate an improvement in the rate of
loss of VC in other progressive neuromuscular conditions such as ALS, as
a result of NIV initiation \textsuperscript{34}. The introduction of
daytime NIV (mouthpiece ventilation) in DMD patients has been
demonstrated to increase the endurance of fatigued inspiratory
muscles\textsuperscript{35}. In a 3-month prospective controlled trial,
Schonhofer et al. demonstrated a significant increase in inspiratory
threshold load endurance time of 278 +/- 269\% indicating improved
inspiratory muscle function \textsuperscript{36}.

Nickol et al \textsuperscript{37} studied 12 subjects with neuromuscular
disease (NMD, not specified), baseline mean TLC 62\%pred and another 8
with scoliosis, after three months of NIV and found a significant
increase in maximum expired pressure (MEP) and hypercapneic ventilatory
response (HCVR) as well as a trend toward increases in inspiratory
strength indicated by more negative maximum inspiratory esophageal
(SnPes), sniff nasal inspiratory pressure (SNIP). No significant
increase was noted in maximum inspiratory pressure (MIP). Given the
small number of subjects, lack of significance in respiratory muscle
function could have been as a result of a beta error.

\textbf{Mechanical In-Exsufflation (MI-E).} Mechanical cough assistance,
generally administered through a noninvasive mask at pressures of +/- 40
cmH2O or greater, has not only been associated with reduced
hospitalizations and intubations due to more effective airway
clearance\textsuperscript{38} but has also been demonstrated to improve
vital capacity and lung mechanics.

In a study of 21 patients with NMD, 10 of whom had DMD (VC 11\%Pred, all
using NIV) Stehling et al demonstrated an average improvement of 28\% in
VC, from 0.48 +/- 0.15 to 0.59 +/- 0.25 liters after one year of twice
daily MI-E therapy \textsuperscript{39}. As average MI-E pressures were
only +25.1+5.3 and - 25.6 + 5.7 cmH2O, a greater effect on VC might be
anticipated with pressures more commonly utilized clinically. Although
these were older individuals (age 20.6 +/- 3.9 yrs) with severe
respiratory muscle weakness, regular use of MI-E clearly has the
capacity to improve measures such as VC\textbf{.}

Although data are limited, taken together, these observations suggest
that standard therapies recommended by international guidelines may have
a significant effect on some of the primary respiratory outcomes being
evaluated in studies of molecular and gene therapies and as such,
respiratory interventions should be well documented and accounted for in
randomization and evaluation.

\textbf{Effect of phenotypic heterogeneity}

Another important factor potentially confounding the interpretation of
aggregate results of intervention studies on respiratory outcomes is
phenotypic heterogeneity. This variability has been recognized for
years\textsuperscript{18} and can manifest as markedly different timing
and value maximum VC as well as rate of decline. Neve, in a study of 33
CS naïve DMD subjects identified subphenotypes requiring NIV either
prior to, or after age 17 \textsuperscript{40}. The former was
identified by an earlier (age 11.5 to 13) and lower peak VC and SNIP and
more rapid decline. In a study using the STRIDE nonsense mutation
(nmDMD) cohort, investigators found no correlation in 181 patients
between disease severity and exon location of the nonsense mutation.
There was a large degree of phenotypic variability despite of having
identical dystrophin mutations \textsuperscript{41}. Birnkrant has
identified highly discordant cardiopulmonary function in brothers with
DMD and identical dystrophin mutations and identified 8 of 15 patients
(53\%) with a shared genotype who had discordant cardiopulmonary
function\textsuperscript{23,42}. Clinical manifestations were not
related to the dystrophin mutation. In a cohort of over 200 patients,
Magri, as well, noted the absence of correlation of pulmonary, cardiac
or motor phenotype with dystrophin genotype, with the exception of
neurocognitive function being related to more distal mutations
\textsuperscript{43}.

Being unable to predict the natural evolution of cardiopulmonary
function in a portion of the population despite common genotype could
pose a significant confounding issue when assessing the impact of
emerging therapies in DMD. Rather than being the true treatment effect,
such beneficial or adverse observations might simply be due to more
favorable or unfavorable phenotype.

\textbf{Results of current gene modifying and molecular therapies}

There is a number of recent studies of novel therapies for DMD
(Ataluren, Indebadone and Etiplersin) which include either primary or
secondary respiratory outcomes.

Ataluren is an oral treatment for patients with nonsense mutation DMD
(nmDMD), designed to facilitate ribosomal readthrough of an in-frame
premature stop codon, increasing production of full-length dystrophin
protein \textsuperscript{44}. Propensity matching, using age at first
symptoms, age at first use and duration of steroid use as well as
time-to-event motor, pulmonary and cardiac features, was employed in
order to match patients in the Strategic Targeting of Registries and
International Database of Excellence (STRIDE) to individuals in an
historical registry, the Cooperative International Neuromuscular
Research Group Duchenne Natural History Study (CINRG
DNHS)\textsuperscript{41}. Individuals were matched according to age at
first symptoms, age at first use and duration of steroid use, as well as
time-to-event motor, pulmonary and cardiac features. Mercuri et al.
demonstrated significant impacts on measures of skeletal muscle function
(standing, stair climbing, timed motor tasks) in nonsense mutation DMD
(nmDMD) but for a number of reasons, including the insensitivity of the
outcome measures used (VC\%pred), the relative youth of the treated
subjects compared to controls and the duration of follow up, no
significant impact on respiratory outcomes was identified.

In a publication describing the results of three studies of Eteplirsin,
Khan \textsuperscript{45} found a significant difference in the rate of
decline of VC\%pred in 74 DMD patients; 2.19 to 3.79 \% per year for
Eteplirsin compared to 5.56 to 6\% per year for the three CINRG DNHS
control groups. However, the study group 201/202, randomized initially
to placebo vs. 30 or 50 mg Eteplirsen, was only 1.5 years younger and
had the greatest improvement in VC decline. However, there was a
considerably higher baseline \%pred VC of 96.9\% for the Eteplirsin
treated subjects vs. at best, 79.6\% for the controls. Interestingly, no
data were presented for the placebo-controlled portion of the study. The
authors concluded that the results were ``meaningful'' and ``will likely
impact both the quality of life and the duration of life in patients
with DMD.''

In a Phase II randomized placebo-controlled study in 21 DMD boys at age
8--16 years, Buyse \textsuperscript{46} found that idebenone, a
short-chain benzoquinone, antioxidant and inhibitor of lipid
peroxidation, at a dose of 450 mg/d had a significant effect on their
primary outcome, peak expired flow (PEF) but only in steroid naïve
subjects. PEF, in the absence of air flow limitation, is not only a
composite reflection of VC, MIP and MEP but also bears a marked clinical
significance in terms of cough effectiveness and airway clearance in
patients with NMD. In DMD, absolute PEF remains quite stable during the
first and second decade but PEF\%pred declines linearly from age 7 or
8\textsuperscript{13}. Not surprisingly, values of PEF\%p correlated
well with the percent predicted values for MIP and FVC. Idebenone was
associated with an 8.0\% improvement in PEF\%pred, while those on
placebo declined by 12.3\% (p \textless{} 0.05) over 12 months.
Indebenone did not significantly affect the rate of decline in steroid
treated subjects.

A subsequent, one-year phase 3 placebo-controlled trial for DMD subjects
age 10-18 yrs who were not using steroids, was also reported. Subjects
were randomized to Idebenone 900 mg daily vs. placebo with the primary
outcome, again being PEF\%pred \textsuperscript{47}. In the intention to
treat analysis, the one-year change in PEF\%pred from baseline was
--2·57 vs. -- 8·84, p\textless{}0·0001. The Idebenone group was 1.5 yrs
younger and although post hoc analysis did not indicate that age had an
effect, younger patients did seem to benefit more, possibly because
their VC (absolute) values were still rising. Observations were not
influenced by past steroid use. No significant differences were found in
the change from baseline for MIP, MEP or peak cough flow. The authors
also noted fewer patients in the treated group reaching milestones of VC
(1 L) and peak cough flow (160 L/min) suggesting that delaying these
thresholds might result in less morbidity and mortality. Avoiding the
need for ventilatory support and airway clearance for a period of time
might indeed be a useful outcome but again, noninvasive ventilation when
needed improves sleep quality and QoL and it is well established that
DMD patients can survive for decades with vital capacities well below
1L, even zero (23, 28, 39).

Taken together, these studies do suggest a modest impact of emerging
therapies on respiratory function decline in individuals with DMD.
Whether or not an improvement in health-related quality of life can be
demonstrated remains to be seen. As long as noninvasive ventilation and
airway clearance strategies are properly employed, survival is unlikely
to be impacted by additional treatments. Mortality continues to be
defined by favorable or unfavorable cardiac function, not respiratory
decline \textsuperscript{48}.

\textbf{Respiratory Outcome Measures to Consider} \textbf{in Future
Studies}

Given the limitations of pulmonary function measures in a number of
studies to date, more sensitive measures are needed which account for
the natural evolution of respiratory function at different ages.

Maximum inspiratory pressure (MIP)

Based on the pressure/volume relationship of the respiratory system and
the reserve present in respiratory muscles, it is accepted that MIP is a
much more sensitive, earlier measure of respiratory muscle impairment
than VC \textsuperscript{49,50}, although VC has been widely used in
clinical management and study outcomes. Indeed, in younger patients, VC
is still rising in to early teen years. In a review paper, Schoser found
MIP included as a secondary outcome in one published\textsuperscript{46}
and three ongoing trials in DMD. In the Idebenone trial
\textsuperscript{46} MIP improved, though not significantly compared to
a decline in the placebo group. There is however contradiction in
published results. Neve studied 33 steroid naïve DMD age 11 (5-16.7 yrs)
and found no significant change in absolute MIP values
\textsuperscript{40} while Gayraud found a declining MIP in a similar
age group (9.1 +/- 1 to 16 +/- 1.4 yrs) with \%pred MIP being only 67\%
at first measurement \textsuperscript{17}. MIP might, then, be superior
to some outcome measures for interventions directed at young patients
with DMD in whom VC is relatively well preserved.

Sniff nasal inspiratory pressure (SNIP)

In the absence of much change in MIP, Neve concluded that sniff nasal
inspiratory pressure (SNIP) was a more sensitive measure of
weakness\textsuperscript{40}. In a 3-year study of 33 steroid-naïve,
5--20-year-old DMD patients, SNIP measurements were found to be
reliable. SNIP and VC increased until 10.5 and 12.5 years of age,
respectively, and then declined. SNIP identified respiratory muscle
impairment earlier (at 10.5 years) than VC (at 12.5 years). Others have
found measurements of SNIP to have less variability and to be performed
consistently in children as young as 5 yrs \textsuperscript{51}.
Standardized performance of SNIP measurements is not entirely agreed
upon and a recent publication, including about 20\% muscular dystrophy
patients, noted that occluding the opposite nostril from that with the
pressure monitor resulted in consistently higher
values\textsuperscript{52}.

Abdominal/Rib Cage contribution to tidal volume

In a retrospective but elegant, 7-year study of 115 individuals with
DMD, 6-24 yrs old, with 574 visits, LoMauro \textsuperscript{13}employed
nonlinear regression model analysis and presented the evolution of mean
curves of spirometry, lung volumes, spontaneous breathing and
thoraco-abdominal pattern, measured by optoelectronic plethysmography.
Although more appropriate for older subjects, measures of respiratory
pattern have the advantage of requiring no cooperation or effort. During
spontaneous breathing it was determined that the abdominal contribution
to tidal breathing was reduced at 14.8 yrs., the tidal volume was
reduced at 17.3 yrs., the minute ventilation was reduced at 18.1 yrs.
and minute ventilation at 22 yrs. Spontaneous respiratory patterns are
not commonly studied but may have a role to play in assessing the effect
of therapeutic interventions, particularly for those who are unable to
cooperate with pulmonary functions measurements such as those under 6
yrs. or with severe weakness or cognitive delay

\textbf{Challenges in Trial design}

As discussed above, the interpretation of the impact of emerging
therapies on respiratory function using aggregate data is confounded by
phenotypic variability where it may not be possible to determine if a
subject is evolving naturally with favorable or unfavorable respiratory
function. As well, the age group commonly evaluated combines subjects
whose VC may be rising with others whose VC is declining. When true
randomization is not possible, attempts to accommodate for this have
included propensity matching as in the ataluren
study\textsuperscript{41}. However, propensity score matching can only
adjust for measured covariates, and one cannot be assured that all
confounding factors have been eliminated \textsuperscript{52}.

Becuase of the somewhat unpredictable evolution of respiratory function
in any given individual which may confound study results for a treatment
intervention, consideration might be given to an N of 1, crossover
design where subjects are compared with themselves under control and
experimental conditions and crossed over at intervals from one to the
other \textsuperscript{53}. The duration of recent studies and the
suggestion of improvements due to treatment as early as 26
weeks\textsuperscript{47}, indicate that these investigations might lend
themselves to such an N of 1 design, e.g. randomization to 6 to 12
months of intervention or placebo, followed by crossover for the
remaining 6 to 12 months. Such a design would help address the issues
with regard to randomization and phenotypic variability.

Future studies should employ outcome measures which take in to account
the dynamic of respiratory function evolution at different ages and
which are sensitive to change. A number of parameters should be followed
including those which are sensitive to respiratory muscle strength (MIP,
SNIP) and absolute VC which, particularly in an N of 1 design, would
account for individual variations in age at maximal VC and rate of
decline. The utilization and impact of standard therapies must also be
considered in fully understanding the effect of emerging therapies.

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