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\begin{document}

\title{Overview of Gene Therapy in Spinal Muscular Atrophy and Duchenne
Muscular Dystrophy}



\author[1]{Nicolas Abreu}%
\author[1]{Megan Waldrop}%
\affil[1]{Nationwide Children's Hospital}%


\vspace{-1em}



  
  \date{May 07, 2024}


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\selectlanguage{english}
\begin{abstract}
Both 5q-linked spinal muscular atrophy (SMA) and Duchenne muscular
dystrophy (DMD) are fatal monogenic neuromuscular disorders caused by
loss-of-function mutations. SMA is an autosomal recessive disorder
affecting motor neurons that is typically caused by homozygous
whole-gene deletions of SMN1. DMD is an X-linked recessive muscle
disease most often due to exon deletions, but also duplications and
smaller sized variants within the DMD gene. Gene replacement therapy
offers the opportunity to correct the underlying genetic defect by the
introduction of a functional gene. We review the transformative work
from clinical trials to United States Food and Drug Administration
approval of onasemnogene abeparvovec-xioi in SMA and its application in
clinical practice and the early results of microdystrophin delivery in
DMD. We also review the introduction of antisense oligonucleotides to
alter pre-mRNA splicing to promote exon inclusion (as in nusinersen in
SMA) or exclusion (as in eteplirsen in DMD) into neuromuscular
therapeutics. There are multiple promising novel genetically mediated
therapies on the horizon, which in aggregate point towards a hopeful
future for individuals with SMA and DMD.%
\end{abstract}%



\sloppy


Abstract

Both 5q-linked spinal muscular atrophy (SMA) and Duchenne muscular
dystrophy (DMD) are fatal monogenic neuromuscular disorders caused by
loss-of-function mutations. SMA is an autosomal recessive disorder
affecting motor neurons that is typically caused by homozygous
whole-gene deletions of \emph{SMN1} . DMD is an X-linked recessive
muscle disease most often due to exon deletions, but also duplications
and smaller sized variants within the \emph{DMD} gene. Gene replacement
therapy offers the opportunity to correct the underlying genetic defect
by the introduction of a functional gene. We review the transformative
work from clinical trials to United States Food and Drug Administration
approval of onasemnogene abeparvovec-xioi in SMA and its application in
clinical practice and the early results of microdystrophin delivery in
DMD. We also review the introduction of antisense oligonucleotides to
alter pre-mRNA splicing to promote exon inclusion (as in nusinersen in
SMA) or exclusion (as in eteplirsen in DMD) into neuromuscular
therapeutics. There are multiple promising novel genetically mediated
therapies on the horizon, which in aggregate point towards a hopeful
future for individuals with SMA and DMD.

Manuscript

Introduction

Gene therapy provides a new avenue to treat life-limiting neuromuscular
disorders. For single-gene conditions resulting from absent or reduced
gene expression, as in spinal muscular atrophy (SMA) and Duchenne
muscular dystrophy (DMD), the goal of gene replacement therapy is to
deliver an intact copy of the disrupted gene, known as a transgene, to
cells so they can express a functional protein to alleviate disease.
This has been performed \emph{ex vivo} for hematological and
immunological disorders by removing hematopoietic stem and progenitor
cells from affected individuals and introducing a transgene into the
extracted cells typically using a viral vector in a tissue
culture.\textsuperscript{1} The goal is to integrate the transgene into
the host genome so that when the treated cells are reintroduced into the
patient, they transfer the transgene onto their daughter cells after
engraftment. In contrast, gene therapy for neuromuscular conditions
requires transduction of long-lived, non-replicating nerve and slower
replicating muscle cells, and so \emph{in vivo} gene therapy strategies
are preferred. Here, a transgene is packaged within a viral vector and
delivered directly into an affected individual, where the viral
machinery allows the transgene to enter cells and replicate without need
to integrate into the host genome.\textsuperscript{1}

There are several considerations for \emph{in vivo} gene replacement
therapy in neuromuscular disease. One advantage of this method is the
lack of integration of the transgene, which has been shown in some cases
to result in unwanted mutations and an increased risk of
cancer.\textsuperscript{2} A major advance has been the development of
adeno-associated virus (AAV) vectors, which are not known to be
associated with human disease and only weakly activate the innate immune
system.\textsuperscript{3,4} There are many AAV serotypes that differ in
terms of tissue tropism and prevalence of pre-existing antibodies in the
general population, leading AAV9 to be a common vector in neuromuscular
gene therapy due to its ability to efficiently transduce the nervous
system and muscle, and lower prevalence of high titers of pre-existing
neutralizing AAV9 antibodies in the community.\textsuperscript{5,6} A
limitation to the use of an AAV vector though is its small carrying
capacity of 5 kilobases, which is much smaller than the
\emph{DMD}gene.\textsuperscript{7}

Another step towards progress in neuromuscular therapeutics was the
introduction of antisense oligonucleotides (ASO). Distinct from gene
therapy, there is no introduction of foreign genetic material to
generate novel transgene expression, but instead this is an RNA-based
therapeutic strategy to alter expression of existing genes. ASOs are
short sequences of modified synthetic nucleotides that bind to specific
sites within pre-mRNA to alter splicing based on their design, and
thereby change exon number in the mature transcript to produce a more
functional protein. They may also be engineered to promote degradation
of the transcript in diseases that result from toxic gain-of-function
mutations, as in Huntington disease, but are not applicable in disease
with loss of protein function like SMA and DMD.\textsuperscript{8}

In this review, we highlight the tremendous progress in genetically
mediated therapies for SMA and DMD, with a focus on ASOs and gene
replacement therapy. We explore some exciting novel therapeutics using
alternative strategies as well in order to show the bright future ahead
for neuromuscular medicine.

\begin{enumerate}
\tightlist
\item
  Gene Therapy in Spinal Muscular Atrophy
\item
  Understanding Spinal Muscular Atrophy
\end{enumerate}

Spinal muscular atrophy (SMA) results from degeneration of the anterior
horn cells of the spinal cord and motor nuclei in the lower brainstem,
and is associated with progressive, symmetric, proximal more than distal
muscle weakness and atrophy. The most common form is due to biallelic
loss-of-function variants in \emph{SMN1} (survival motor neuron 1 gene).
Occurring in about 8 in 100,000 live births, it has been the leading
inherited cause of infant death.\textsuperscript{9} \emph{SMN1} is
located on the long arm of chromosome 5, specifically the 5q13.2 region,
and thus \emph{SMN1-} related SMA may also be referred to as 5q SMA.
About 94\% of individuals with \emph{SMN1-} related SMA carry homozygous
deletions of exon 7, though alleles with smaller deletions, nonsense
variants, frameshift variants, splice site mutations, and missense
variants have all been described.\textsuperscript{10} There is a group
of disorders known as non-5q spinal muscular atrophies that may mimic
the clinical appearance to \emph{SMN1-} related disease, but are due
to many other genetic etiologies and often on close evaluation have
distinguishing features from typical SMA.\textsuperscript{11} Hereafter,
the use of SMA will exclusively refer to 5q spinal muscular atrophy.

Individual phenotypes of SMA are largely driven by copy number of a
modifying gene, \emph{SMN2} . This gene is nearly identical
to\emph{SMN1} , with a unique C to T transition in exon 7 that results
in exclusion of exon 7 during post-transcriptional processing, leading
to the production of mostly nonfunctional SMN protein. However, 10-15\%
of mRNAs from \emph{SMN2} retain exon 7 and produce some full-length SMN
protein.\textsuperscript{12,13} The phenotypic severity is inversely
related to the number of copies of \emph{SMN2} present, where
infantile-onset SMA is typically associated with 2 copies of\emph{SMN2}
, while adult-onset SMA is typically associated with 4 or more copies of
\emph{SMN2.} \textsuperscript{10,14,15}

Prior to the onset of disease-modifying therapies, SMA was categorized
into types based upon age and severity of presentation. Children with
Type 0 SMA typically have 1 copy of \emph{SMN2} and have neonatal onset
of symptoms, historically dying well within the first 6 months of life
from respiratory complications. Type 1 SMA is the most common of the
subtypes and has been classified as disease onset after birth but prior
to 6 months of age, with inability to sit
unassisted.\textsuperscript{16} Children with Type 1 SMA have 2-3 copies
of \emph{SMN2} and have a life expectancy of about 2 years of age due to
respiratory failure prior to directed
treatments.\textsuperscript{17,18}Type 2 SMA is a more attenuated form,
with symptom onset in later infancy or toddlerhood, being able to sit
but not able to walk. They often have 3 copies of SMN2, and eventually
lose the ability to sit unsupported, but two-thirds live through their
mid-twenties.\textsuperscript{19} Type 3 refers to individuals who
achieve the ability to walk but then lose that skill, with 3-4 copies
of\emph{SMN2} , and associated with a normal lifespan. For Type 4 SMA,
with 4 or more copies of \emph{SMN2} , symptoms begin after 30 years of
age, and individuals typically continue to ambulate and have a normal
life expectancy.\textsuperscript{19}

Landscape of non-gene replacement therapy treatment options for Spinal
Muscular Atrophy

Prior to 2016, there were no approved disease-modifying therapies in the
United States for SMA. Historically, the mainstay of care focused on
management of complications of the disease and improving quality of
life. Supporting respiratory function, nutritional and gastrointestinal
status, bone health, mobility, neurodevelopment, and psychosocial
wellness were and remain cornerstones of comprehensive spinal muscular
atrophy care.\textsuperscript{20,21}

The first medication approved by the U.S. Food and Drug Administration
(FDA) for the treatment of SMA was nusinersen, a 2-methoxyethyl modified
ASO designed to target the intronic splicing silencer N1 to suppress the
routine splicing out of exon 7 from \emph{SMN2} pre-mRNA (Table 1). By
displacing the hnRNP proteins at the site, nusinersen increases the
synthesis of transcripts containing exon 7 and ultimately full-length
SMN protein.\textsuperscript{22} In a multicenter phase 3 randomized,
double-blind, sham-controlled trial of nusinersen in infants with Type 1
SMA treated at a mean age of 7.9 weeks, the trial terminated early due
to a high percentage (41\%) of treated infants showing improvement in
motor milestones on Section 2 of the Hammersmith Infant Neurological
Examination, compared to 0\% in the control group. In the final
analysis, 51\% of treated infants showed a motor milestone response and
an overall increased likelihood of survival over 13
months.\textsuperscript{23} This work was followed by a phase 3 trial of
Type 2 SMA children aged 2-9 years. After 15 months of treatment, there
was an increase in \textasciitilde{}4 points on the Hammersmith
Functional Motor Scale-Expanded compared to a decrease in
\textasciitilde{}1 point in the control group, resulting in early
termination. Though, there was no significant difference between groups
in the proportion of children who were able to stand alone or walk with
assistance.\textsuperscript{24}

For adults with Type 3 SMA treated between 18 and 59 years of age with
nusinersen, they were able to walk further by a modest amount over 10
months of treatment when compared to baseline on the 6-minute walk test
(mean 369.5 meters v. 377.75 at final
visit).\textsuperscript{25}Nusinersen was approved by the FDA for all
types of SMA and is administered intrathecally as the ASO cannot cross
the blood-brain barrier. It is a life-long treatment, requiring three
lumbar punctures per year in the maintenance phase after completion of
four loading doses. Monitoring for thrombocytopenia, coagulation
abnormalities and renal toxicity is recommended at baseline and prior to
each dose. It is overall a safe and well tolerated treatment, though
sedation may be necessary in young children or those with procedural
anxiety, and advanced surgical planning may be needed in those
considering spinal fusion for scoliosis.\textsuperscript{26}

An emerging alternative to nusinersen is the orally bioavailable small
molecule, risdiplam. It is in a different class of therapeutics to ASOs
as risdiplam may be given enterally in liquid form daily instead of
requiring intrathecal administration.\textsuperscript{27} Risdiplam is a
centrally and peripherally distributed splicing modifier that binds to
exon 7 of \emph{SMN2} pre-mRNA, displacing hnRNP proteins and promoting
inclusion of exon 7 for production of full-length SMN
protein.\textsuperscript{27,28} Risdiplam has shown to be safe in
healthy volunteers despite concerns of off-target effects based on a
related splicing modifier RG7800 demonstrating retinal toxicity in
cynomolgus monkeys.\textsuperscript{29} Based on interim analyses of
FIREFISH (NCT02913482), 93\% of SMA Type 1 infants aged 1-7 months had
at least a 4 point improvement in motor functioning from baseline on the
total score of the Children's Hospital of Philadelphia Infant Test of
Neuromuscular Disorders (CHOP-INTEND) after 16 months of
treatment.\textsuperscript{30} In addition, improvement in motor
functioning has been suggested for treated individuals with Type 2 and
Type 3 SMA ranging in age from 2 to 25 years old in a multicenter,
double-blind, placebo-controlled study (SUNFISH,
NCT02908685).\textsuperscript{31} As of June 2020, risdiplam remains
under priority review with the FDA.

Gene Replacement Therapy for children with Spinal Muscular Atrophy

In May 2019, onasemnogene abeparvovec-xioi became the first \emph{in
vivo} systemically delivered gene therapy approved by the FDA (Table 1).
The treatment involves a one-time intravenous (IV) administration
of\emph{SMN} complementary DNA, from which full-length SMN protein may
be produced. The transgene is under the control of a cytomegalovirus
enhancer/chicken-beta-actin hybrid promoter within a self-complementary
adeno-associated viral serotype 9 (scAAV9) to drive high levels of gene
expression.\textsuperscript{32} This serotype of AAV is ideal due to its
ability to cross the blood-brain barrier and its robust transduction of
brain and spinal cord neurons, including alpha motor neurons known to
underlie the pathogenesis of SMA.\textsuperscript{33} The engineering of
a self-complementary template allows the coding region of the
recombinant virus to form an intramolecular double-stranded DNA,
allowing for efficient replication and transcription rather than waiting
for host cell-mediated synthesis of the second
strand.\textsuperscript{34} One-time administration is possible based
upon the longevity and non-replicating nature of motor neurons.

In an SMA mouse model with a typical lifespan of 13 days, gene
replacement using 5x10e11 genomes demonstrated excellent motor outcomes
and survival was extended from a median age of 15.5 days in a group
treated with scAAV9 without SMN to more than 250 days in the treated
cohort with scAAV9-SMN when administered intravascularly on postnatal
day 1.\textsuperscript{32,35} There was partial improvement in survival
when mice were treated at postnatal day 5, and little effect at
postnatal day 10, which may be related to physiologic postnatal
astrocyte genesis and associated trapping of AAV9 in
mice.\textsuperscript{36}

In the landmark phase 1/2a, open-label, dose escalation trial of
onasemnogene abeparvovec of 15 Type 1 SMA children with two copies
of\emph{SMN2} treated between 0.9 and 7.9 months of age, there was
marked improvement in motor milestones and all had event-free survival
(ventilatory support \textless{} 16 hours per day) compared to 8\% of
natural history controls at 20 months of age. Of the 12 individuals in
the high-dose cohort (2x10e14 vector genomes/kilogram of body weight),
11 were able to sit unassisted for at least 5 seconds, 9 for at least 30
seconds, 9 could roll over, and 2 were able to crawl and walk
independently.\textsuperscript{37}

After the first patient was noted to have significantly elevated serum
aminotransferase levels, alanine aminotransferase (ALT) 31X ULN and
aspartate aminotransferase (AST) 14X ULN but without clinical
manifestations, subsequent patients were immunosuppressed with
prednisolone 1 mg/kg for 30 days starting 1 day prior to gene transfer.
Prednisolone attenuated this response, with only three individuals in
the high-dose cohort showing elevations in AST and ALT. Two had
elevations of less than 10X ULN, and one individual had elevations that
required additional prednisolone as a result of ALT at 35X ULN and AST
at 37X ULN.\textsuperscript{37} All aminotransferase elevations were
transient and asymptomatic.

Follow-up for an additional two years post-gene transfer revealed two
additional patients achieved independent sitting for at least 30 seconds
and two others achieved standing with support.\textsuperscript{38} In
addition, the majority (11/12) of children tolerated at least partial
oral feeding safely and spoke by the end of long-term follow-up. Dosing
earlier than three months of age may be associated with greater
improvement in motor outcomes, and to this point, the oldest patient in
the trial at 7.9 months (also with low baseline motor functioning)
demonstrated only modest improvement in functioning.\textsuperscript{38}

Phase 3 trials are underway in the United States, Europe, and East Asia,
as well as trials exploring pre-symptomatic treatment and intrathecal
onasemnogene abeparvovec-xioi administration for individuals with
more\emph{SMN2} copies (NCT03505099, NCT03306277, NCT03461289,
NCT03837184, NCT03381729), with encouraging interim
results.\textsuperscript{39} As of June 2020, the open-label phase 1/2a
trial of intrathecal onasemnogene abeparvovec remains on a partial hold
by the FDA after review of non-human primate data demonstrating
inflammation of the dorsal root ganglia, which has not been seen in any
humans who had already received intrathecal gene
transfer.\textsuperscript{40}

The clinical practice of gene therapy for Spinal Muscular Atrophy

Newborn screening for SMA has revolutionized care, allowing early
identification of SMA and thus maximizing response to gene
transfer.\textsuperscript{41--43} While SMA was included in the
recommended U.S. universal screening program for newborn screening in
2018, implementation is slow and is currently active in only a minority
of states.\textsuperscript{44} While initial clinical trials of gene
replacement therapy targeting young infants with SMA with 2 copies
of\emph{SMN2} , FDA approval for onasemnogene abeparvovec-xioi was
granted for all children with SMA less than 2 years of age without
end-stage disease.\textsuperscript{5}

Once an eligible individual is identified via newborn screen or in
clinic, confirmatory SMA genetic testing with \emph{SMN2} copy number
should be completed (Figure 1). Once the genetic diagnosis is confirmed,
a safety evaluation with AAV9 titers and baseline ``SMA gene transfer
laboratories'' (ALT, AST, bilirubin, prothrombin time, platelet counts,
as well as consideration of human immunodeficiency virus (HIV),
hepatitis B and C serologies) are essential prior to onasemnogene
abeparvovec administration.\textsuperscript{45} Elevated AAV9 titers may
increase risk of immunogenicity and limit efficacy of treatment, and so
individuals with anti-AAV9 antibody titers greater than 1:50 have been
excluded from intervention.\textsuperscript{45} The prescribing
information also recommends monitoring troponin I levels at baseline and
after treatment to monitor for cardiac effects, although no clinical
cardiac toxicity has been noted.\textsuperscript{37}

Onasemnogene abeparvovec is given via IV infusion over 60 minutes at a
dose of 1.1 x 10e14 vg/kg.\textsuperscript{37,45} Prednisolone 1
mg/kg/day begins one day prior to gene transfer, and is recommended for
at least 30 days prior to weaning, with adjustment in dosing depending
on ongoing monitoring; in some instances, the prednisolone dose may need
to be increased to further suppress the AAV9 immune
response.\textsuperscript{37} Weekly laboratory monitoring should
continue until AST and ALT are normalizing (\textless{}2X ULN) and then
may be spaced out. The most common side effects include vomiting and
elevated aminotransferases. Onasemnogene abeparvovec-xioi carries a
black box warning of acute liver injury after an individual with Type 1
SMA with premorbid elevated aminotransferases developed acute liver
injury with jaundice, AST 80X ULN and ALT 45X ULN, and massive
inflammation of liver biopsy 7 weeks after treatment administration
under the managed access program.\textsuperscript{45} The child returned
to baseline status after use of corticosteroids and had already
completed the recommended 30 days of 1 mg/kg of prednisolone and a
2-week taper. There is also description of transient decreases in
platelet counts and elevations in cardiac troponin-I levels of unclear
clinical significance.\textsuperscript{45} Overall, onasemnogene
abeparvovec is a safe and highly effective treatment when care is given
to treat children with no underlying liver dysfunction, and prednisolone
is adjusted based upon close monitoring throughout the immediate
post-treatment period.

Although the viral vector for onasemnogene abeparvovec-xioi does not
cause human disease, guardians and childcare providers of infants who
undergo gene transfer are counseled on use of protective gloves when
coming into direct contact with patient bodily fluids and waste, as well
as good hand hygiene for 4-6 weeks after administration to minimize
exposures as a result of viral shedding. If exposed, caregivers may
develop AAV9 immunity precluding them from future potential AAV9
therapies. Breastfeeding was restricted in the phase 1 trial before,
during, and 30 days after gene transfer out of the theoretical risk of
passive maternal transfer of anti-AAV9 antibodies, however the risks and
benefits should be weighed on an individual basis with the treating
physician.\textsuperscript{5,37,45} Vaccinations may be adjusted at the
discretion of the treating physician and may be given at least one week
prior to gene transfer. Inactivated vaccinations may be given at any
time post gene transfer, but live attenuated vaccines should not be
given until 4 weeks after completing the prednisolone
course.\textsuperscript{5,37,46,47}

\begin{enumerate}
\tightlist
\item
  Gene Therapy in Duchenne Muscular Dystrophy
\item
  Understanding Duchenne Muscular Dystrophy
\end{enumerate}

Duchenne muscular dystrophy (DMD) is on the severe end of the spectrum
of X-linked dystrophinopathies, which are life-limiting muscle diseases
associated with mutations in the dystrophin-encoding gene, \emph{DMD} .
DMD is characterized by loss of ambulation prior to age 13 while the
milder form, Becker muscular dystrophy, has a wide phenotypic spectrum
with loss of ambulation after 16 years or not at all. Dystrophin is
located on the cytoplasmic side of the muscle fiber plasma membrane,
providing structural integrity to the cell when bound to the associated
transmembrane glycoprotein complex. Absence of dystrophin causes muscle
membrane instability, leading to degeneration with impaired
regeneration, fibrosis and fatty infiltration in
muscle.\textsuperscript{48}

DMD is identified in about 1 in 5000 to 6000 live male
births.\textsuperscript{49,50} Although males are mostly affected as
this is an X-linked recessive disorder, symptomatic female carriers have
been described with variable age of onset and severity of
symptoms.\textsuperscript{51} Early symptoms of DMD include delayed
gross motor milestones, calf enlargement, a waddling gait, toe-walking,
and a Gower's maneuver. Symptoms are first noticed \textasciitilde{}2-3
years of age, though significant delays in diagnosis
occur.\textsuperscript{52} Diagnosis is confirmed with
\emph{DMD}deletion and duplication analysis and sequencing, though in
very rare instances muscle mRNA analysis may be
needed.\textsuperscript{53}

Approximately 60-70\% of dystrophinopathies result from deletions of one
or more exons within the \emph{DMD} gene.\textsuperscript{54,55} Less
common mutations include intragenic duplications, single nucleotide
variants, splice site changes, and deletions and insertions of a few
nucleotides. Clinicians typically use the reading frame rule to assist
in phenotype predictions. Deletions that result in a shift in the mRNA
translational reading frame (out-of-frame deletions) lead to very
minimal or absent dystrophin production and a more severe DMD phenotype.
Deletions that preserve the translational reading frame (in-frame
deletions) lead to incomplete dystrophin production with some residual
function and are predicted to lead to a milder BMD phenotype. About 90\%
of individuals with DMD due to intragenic deletions are out-of-frame,
though this is not as consistently seen in BMD with in-frame
deletions.\textsuperscript{56--58}

DMD results in multisystem dysfunction as a result of the underlying
muscle disease, as well as adverse effects from long-term enteral
corticosteroids used as a part of current standard of care.
DMD-associated cardiomyopathy and conduction defects will be explored in
more detail in other articles in this special edition, as well as the
respiratory complications from progressive loss of strength of
respiratory and bulbar muscles. Gastrointestinal function is also
affected, with constipation, reflux and delayed gastric emptying that in
part may be related to altered function of dystrophin in smooth muscle
in the gastrointestinal tract. Short stature, obesity and low bone
mineral density are common and may be complicated by corticosteroid use.
Orthopedic sequelae include contractures and scoliosis.
Neurodevelopmental disorders like intellectual disability, learning
disabilities, autism spectrum disorder and attention-deficit
hyperactivity disorder, are seen at higher rates compared to the general
population. \textsuperscript{59,60} Further, patients and their
caregivers are at increased risk of depression and anxiety.
Multidisciplinary muscular dystrophy care is vital to address the many
needs of these individuals and ensure the best possible
outcomes.\textsuperscript{61--63} Although advances like scoliosis
surgery and non-invasive ventilation have increased the median survival
of individuals with DMD to \textasciitilde{}30 years, the importance of
disease-directed therapies remains.\textsuperscript{64}

Landscape of non-gene therapy treatment options for Duchenne Muscular
Dystrophy

Glucocorticoids have been the long-standing crux of neuromuscular care
for DMD, first showing benefit in 1974.\textsuperscript{65} Their
specific mechanism of action in DMD and effects in BMD remain unknown.
Prednisone and deflazacort, a corticosteroid prodrug, improve motor
functioning, delay onset of cardiomyopathy, reduce the need for
scoliosis surgery, and may prolong survival (Table
1).\textsuperscript{66,67} Daily corticosteroid use is not without side
effects and alternative dosing regimens have been suggested, including
weekend dosing, which may be equally beneficial for motor outcomes and
have less effects on weight gain or reduction in linear
growth.\textsuperscript{68} Treatment has evolved from initiation at the
start of motor decline, to more recently around 4-5 years of age prior
to decline in motor skills, though there may be benefit to starting at
an even younger age with a weekend dosing
regimen.\textsuperscript{69}Medication typically continues indefinitely
while monitoring for side effects, which may prompt dose reductions.
Weight gain, hirsutism and cushingoid appearance are the most common
side effects, though short stature, delayed puberty, fractures, reflux,
hypertension, cataracts, acne, striae, and behavioral dysregulation all
may occur. Physicians, patients and families must also be aware of the
possible need for stress-dose steroids around the time of illness or
scheduled surgeries in order to prevent adrenal crises.

Despite decades of research on the genetics and spectrum of mutations of
DMD, novel treatments addressing the underlying pathophysiology have
only recently emerged. Exon skipping interventions were the first viable
precision therapies for DMD. Exon skipping relies on the use of
antisense oligonucleotides (ASO) to bind at a specific location of the
DMD pre-mRNA, altering splicing to exclude the exon in the mature
mRNA.\textsuperscript{8} When applied in DMD to deletions that disrupt
the reading frame, masking one exon may restore the reading frame and
result in some expression of shortened but functional dystrophin.

Eteplirsen is a first-generation morpholino ASO, otherwise known as a
phosphorodiamidate morpholino oligomer (PMO), that has modifications to
the ribose sugar to avoid degradation from nucleases and improve target
affinity. It promotes exon 51 exclusion and restores the reading frame
in individuals with amenable mutations (e.g., deletions of exons 48-50
or exon 52). Despite the large number of variants, \textasciitilde{}13\%
of all individuals with DMD bear exon 51 skipping-amenable mutations.
The second and third most applicable sites for single exon skipping are
at exon 45 and 53, making up 8.1\% and 7.7\% of all DMD mutations
respectively.\textsuperscript{70} Eteplirsen is administered by IV
infusion weekly, typically via an implanted central venous catheter.
Clinical trials of the PMO have demonstrated a small but significant
amount of dystrophin expression, attenuation of respiratory decline
based on forced vital capacity, and improved ambulation in the majority
of treated individuals when compared to historical
controls.\textsuperscript{71--75} The FDA approved eteplirsen in
September 2016 based on limited data using the surrogate marker of
dystrophin expression, no adverse events and the expectation of future
clinical benefit (Table 1).\textsuperscript{76}

The second ASO to be approved by the FDA for DMD was the exon
53-skipping PMO, golodirsen (Table 1). With once weekly IV infusions
over 48 weeks, 25 individuals with DMD showed increased dystrophin
expression compared to baseline.\textsuperscript{77} The FDA noted
concerns of renal toxicity seen in animal models treated with a 10X
higher dose than what has been used in humans, as well as risk of
indwelling catheter infections in their initial rejection, requesting
renal function monitoring and demonstration of meaningful change in
functional outcomes in the larger scale phase 3 ESSENCE trial
(NCT02500381) that is currently underway.\textsuperscript{78}

Ataluren is a distinct small molecule therapy administered enterally
that induces ribosomal readthrough of premature stop codons as a result
of \emph{DMD} nonsense mutations, which comprise about 10\% of all DMD
cases (Table 1).\textsuperscript{54,79} While the primary endpoint of
improvement in ambulation was not met in the phase 3 trial, additional
analyses and review of registry data suggest a
benefit.\textsuperscript{80--82} Ataluren has conditional authorization
in Europe by the EMA, but is not approved by the
FDA.\textsuperscript{83,84} There are a number of other small molecule
therapies, exon-skipping technologies, and steroidal therapies currently
under investigation or under FDA review that are beyond the scope of
this review.\textsuperscript{85--87}

Gene transfer for children with Duchenne Muscular Dystrophy

Gene replacement therapy in DMD holds great promise among emerging
therapies by directly providing a functional copy of the \emph{DMD}gene,
addressing the underlying genetic defect regardless of the underlying
mutation. With the discovery of AAV vectors, efficient transgene
transduction in muscle was achieved without the challenges seen with
\emph{in vivo} use of adenovirus or lentivirus
vectors.\textsuperscript{3} However, due to the large size of the
dystrophin gene and small carrying capacity of AAV vectors, researchers
have designed synthetic microdystrophins, removing many of the
spectrin-like repeats and the C-terminal domain to produce a transgene
that may fit within an AAV vector and produce a functional, internally
truncated protein akin to ``in-frame'' BMD-like
mutations.\textsuperscript{3} The first in-human trial of AAV-mediated
gene transfer in DMD was performed in 6 patients via intramuscular
delivery. Safety was established, but transfer resulted in no
significant levels of microdystrophin expression.\textsuperscript{88} In
addition, evidence of immunogenicity was noted with detectable T-cell
responses to the AAV vector and the microdystrophin transgene. This
informed subsequent trials by highlighting challenges with pre-existing
neutralizing antibodies against the vector, poor vector uptake due to
AAV2.5 serotype, and lack of tissue specificity of the CMV early
enhancer plus promoter, which may have boosted the immune
response.\textsuperscript{3,88,89} There are three active IV
microdystrophin trials in the United States with important differences
in AAV serotype, promoter, transgene, and inclusion criteria
(NCT03769116,, NCT03362502, NCT03368742).\textsuperscript{3}

Results from an open-label phase 1/2a trial of systemic microdystrophin
in four children with DMD using an AAVrh74 vector and MHCK7 promoter
revealed high levels of microdystrophin expression 12 weeks after gene
transfer and functional improvements in motor outcome measures sustained
over a 1-year period.\textsuperscript{90} All participants showed a
reduction in creatine kinase levels at 1 year of age (though levels
fluctuated in between), and all scores improved by at least 2 points on
the North Star Ambulatory Assessment compared to baseline. The treatment
was safe, with self-limited vomiting and transient liver enzyme
elevations as the most common adverse effects. A dose of 2.0 x 10e14
vg/kg was used in boys aged 4-6 years and AAVrh74-binding antibodies
were permitted up to 1:400.\textsuperscript{37,90} All children were on
a weekend steroid regimen prior to enrollment, and so an additional 1
mg/kg daily dose of prednisone (or equivalent) was given each weekday
for 30 days and weaned over 2-4 weeks. In this small sample, there were
only modest elevations of liver enzymes, with gamma-glutamyltransferase
peaking at four times ULN. It is not clear if this milder hepatic
response than as was seen with onasemnogene abeparvovec in SMA may be
due to sample size, engineering or specifics of the vector or transgene,
or pre-existing use of steroids as standard-of-care in DMD. A
double-blind, placebo-controlled trial is underway (NCT03769116).

Surrogate gene therapy represents another mutation-independent
therapeutic avenue by delivery of genes via AAV vectors that can
substitute for dystrophin. Utrophin bears much of the structural and
functional elements of dystrophin, and its upregulation via AAV-mediated
gene transfer has been successful at restoring muscle function in animal
models.\textsuperscript{91} Similarly, B4GALNT2 (GALGT2) upregulation
via AAV-mediated delivery results in increased expression of
dystrophin-associated proteins, correction of muscle histopathology, and
improvement in cardiac function in preclinical
studies.\textsuperscript{92--94} Their clinical benefit in children with
DMD remains unknown.

Conclusions

Precision medicine for neuromuscular disorders has transformed in the
past decade with commercially available gene replacement therapy for SMA
and ASOs for both SMA and DMD. Additional small molecule and gene
therapies are currently in development for these and other neuromuscular
conditions, like risdiplam for SMA and gene replacement therapy for DMD
and X-linked myotubular myopathy.\textsuperscript{27,86,95--97} Further,
gene editing using CRISPR/Cas9 systems has been successful in large
animal models in DMD and are progressing in their development toward
human applications.\textsuperscript{98} Yet, as exciting as these
treatments are, clinicians must remain committed to continued
multidisciplinary care of the complications of the underlying
neuromuscular disease as we are learning about the long-term safety and
longevity of these therapies. Efforts to expand gene therapy for SMA for
those of older ages (NCT03381729) are ongoing, along with research on
how to safely and effectively treat those with pre-existing AAV
antibodies.\textsuperscript{99,100} It is unknown if the combination of
gene replacement with other genetically-mediated therapies will provide
added benefit. This requires further evaluation as families and health
care systems are facing the high costs of these
interventions.\textsuperscript{101,102} By fostering collaboration
between researchers, clinicians, patient advocacy groups, government,
and industry, the field will continue to move forward in this new era of
neuromuscular medicine.

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Table 1. Approved medications for spinal muscular atrophy and Duchenne
muscular dystrophy in the United States.

Legend: * - Approved for use in Europe for children with SMA less than
21 kilograms (approximately under 5 years of age) and up to
three\emph{SMN2} copies. \textsuperscript{\#} - Initial approval for
deflazacort for use in children with DMD five years of age and older was
revised to include children who are two years of age and older on June
7, 2019.

Figure 1. Clinical decision-making algorithm for the use of gene
replacement therapy in spinal muscular atrophy.

Legend: \textsuperscript{§} - Evaluation for \emph{SMN2} copy number
should be conducted during genetic confirmation to inform prognosis and
aid in conversations about medical decision-making even though
treatments in the United States are not currently limited by
\emph{SMN2}copy number. \selectlanguage{english}\textsuperscript{+} - Approved for use in Europe
for children with SMA less than 21 kilograms (approximately under 5
years of age) and up to three \emph{SMN2} copies. \selectlanguage{english}\textsuperscript{++} -
One option includes the splicing modifier risdiplam, given as a daily
enteral medication, which is currently available under an expanded
access program (NCT04256265).

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