Seminar
Spinal muscular atrophy
Lancet 2008; 371: 2120–33
Department of Medicine,
Stanford University School of
Medicine, Stanford, CA, USA
(M R Lunn BS); and Department
of Neurology and Neurological
Sciences, Pediatric Neurology
Division, Stanford University
Medical Center, Stanford, CA,
USA (C H Wang MD)
Correspondence to:
Dr Ching H Wang, Department of
Neurology and Neurological
Sciences, Pediatric Neurology
Division, Stanford University
Medical Center, 300 Pasteur
Drive, Room A343, Stanford,
CA 94305-5235, USA
wangch@stanford.edu
2120
Mitchell R Lunn, Ching H Wang
Spinal muscular atrophy is an autosomal recessive neurodegenerative disease characterised by degeneration of spinal
cord motor neurons, atrophy of skeletal muscles, and generalised weakness. It is caused by homozygous disruption
of the survival motor neuron 1 (SMN1) gene by deletion, conversion, or mutation. Although no medical treatment is
available, investigations have elucidated possible mechanisms underlying the molecular pathogenesis of the disease.
Treatment strategies have been developed to use the unique genomic structure of the SMN1 gene region. Several
candidate treatment agents have been identified and are in various stages of development. These and other advances
in medical technology have changed the standard of care for patients with spinal muscular atrophy. In this Seminar,
we provide a comprehensive review that integrates clinical manifestations, molecular pathogenesis, diagnostic
strategy, therapeutic development, and evidence from clinical trials.
Introduction paralysis, and no control of head movement. They are
Spinal muscular atrophy, the leading genetic cause of unable to sit without support. The spared diaphragm,
infant deaths, is an autosomal recessive disease that results combined with weakened intercostal muscles, results in
from degeneration of motor neurons of the spinal cord. paradoxical breathing (inward bony thorax movement
The incidence of spinal muscular atrophy is about one in with outward abdominal movement during inspiration)
10 000 livebirths with a carrier frequency of one in 50.1,2 and a bell-shaped upper torso. Bulbar denervation results
There is no effective medical treatment for spinal muscular in tongue fasciculation and weakness with poor suck and
atrophy. However, since the discovery of the disease-causing swallow. It also decreases airway protection and increases
gene—survival motor neuron 1 (SMN1)—substantial the risk of aspiration pneumonia, an important cause of
progress in understanding the molecular pathogenesis of morbidity and mortality.
this disease has been made. With this knowledge, Type II disease is of intermediate severity and
innovations have generated excitement about the imminent characterised by onset between 7 and 18 months of age.
development of treatment options. These and other Patients are able to maintain a sitting position unaided. A
advancements in medical technology have changed the few are able to stand with leg braces, but none can walk
standard of care for patients with this debilitating disease. independently. Kyphoscoliosis usually develops, requiring
surgical or orthotic intervention. Fine tremors with digit
Clinical manifestations extension or hand grips are also common. Weak
Spinal muscular atrophy is characterised by degeneration swallowing might deter weight gain. Like patients with
of motor neurons of the spinal cord, which results in type I disease, clearing of tracheal secretions and coughing
hypotonia and muscle weakness. Previously, diagnosis might become difficult because of poor bulbar function
was confirmed by electromyography and muscle biopsy. and weak intercostal muscles. Respiratory insufficiency is
Electromyography characteristic of the disorder shows a frequent cause of death during adolescence.
features of denervation with spontaneous activity of Patients with type III spinal muscular atrophy
positive sharp waves, fibrillation, and occasional (Kugelberg-Welander disease) show profound symptom
fasciculations.3,4 Motor unit action potential shows high heterogeneity. They typically reach all major motor
amplitudes and long durations coupled with decreased milestones, such as independent walking. Some might
recruitment. Histopathology of skeletal muscle usually need wheelchair assistance in childhood, whereas others
shows atrophic fibres with islands of group hypertrophy,3 might continue to walk and live productive adult lives
and the spinal cord shows severe loss of motor neuron in with minor muscular weakness. These patients often
the anterior horn region (figure 1). develop scoliosis. Symptoms of joint overuse, generally
Spinal muscular atrophy was previously divided into caused by weakness, are frequently seen. Patients with
three clinical types on the basis of age of onset and motor type IV disease typically have onset of weakness in the
function achieved: (1) severe type I; (2) intermediate type
II; and (3) mild type III.5 Adult-onset type IV has been
added to include very mild disease (table 1).6,7 Most Search strategy and selection criteria
researchers define spinal muscular atrophy type by the We searched for all publications containing “spinal muscular
highest level of motor function—ie, sitter, walker. atrophy”, “SMA”, “survival motor neuron”, and “survival of
Type I disease (Werdnig-Hoffmann disease) is the most motor neuron”. We included all relevant papers after 2000,
severe and common type, which accounts for about but seminal reports, regardless of their publication date, were
50% of patients diagnosed with spinal muscular atrophy.8 also included. We reviewed papers published in English,
It is distinguished by the onset of disease before 6 months German, Italian, Japanese, Russian, and Spanish, but included
of age and death within the first 2 years of life. These only those published in English.
patients have profound hypotonia, symmetrical flaccid
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 Seminar

second or third decade of life. Motor impairment is mild

without respiratory or nutritional problems, and patients A B
are able to walk in adult years. Other forms of spinal
muscular atrophy include X-linked disease, spinal
muscular atrophy with respiratory distress (SMARD),
spinal and bulbar muscular atrophy (Kennedy’s disease),
and distal spinal muscular atrophy, which are beyond the
scope of this Seminar.
Normal SMA

Molecular genetics and pathogenesis

In 1990, investigators used linkage analysis to identify
the locus of the SMA gene on chromosome 5q13.9,10 The
initial 10 cM interval was later narrowed to a 1–2 cM
region with recombinant mapping.11–13 Cloning of the
disease-gene region with yeast artificial chromosomes
identified multicopy repeats of microsatellites (eg, CMS1,
C212, C272) and other genomic sequences (figure 2).
This unstable region was subjected to intrachromosomal
rearrangements, including gene duplications, gene C D
conversions, and de-novo deletions.14–16 This complex
genomic organisation greatly hindered the initial pro-
gress of cloning the disease gene.11,15 In 1994, researchers *
identified deletions of multicopy microsatellites in
patients with spinal muscular atrophy;15 some of which
were in linkage disequilibrium with the disease locus.17
Despite the genomic complexity in this region, Melki and Normal SMA
colleagues15 uncovered a small 11 kb fragment that
uniquely hybridised to a telomeric repeat that was Figure 1: Histopathology of spinal muscular atrophy
Motor commands generated in the cerebral cortex are transmitted through spinal cord α-motor neurons (red cell
missing in patients, thereby further narrowing the in spinal cord anterior horn and green arrow in (A). The spinal cord anterior horn region shows an absence of
candidate region. motor neurons in a patient (B) compared with those in the healthy control (A). Skeletal muscle of a patient (D)
A human fetal brain cDNA library was probed by shows hypertrophic fibres (hollow arrowhead) surrounded by group atrophy (green arrowhead) compared with
genomic DNA from the candidate region, and the SMN healthy fibres with uniform morphology in normal infantile muscle (C). Despite the atrophy of muscle fibres in
spinal muscular atrophy, muscle spindles (black asterisk) are not affected and become more conspicuous (D). All
gene was identified as the disease-causing gene in 1995.18 slides are stained with haematoxylin and eosin.
The SMN gene is present in multiple copies in the human
genome: one SMN1 (SMNT, telomeric) and several SMN2 OMIM Age at onset Highest function Natural age
(SMNC, centromeric). The two genes differ by only five number achieved of death
nucleotides, only one of which is within the 1·7 kb coding Type I (severe, Werdnig-Hoffmann disease) 253300 0–6 months Never sit <2 years
region, but none affect the predicted aminoacid Type II (intermediate) 253550 7–18 months Sit, never stand >2 years
sequence.18,19 Both genes contain nine exons and eight Type III (mild, Kugelberg-Welander disease) 253400 >18 months Stand and walk Adult
introns that span about 20 kb genomic region. SMN1 Type IV (adult) 271150 Second or third Walk during Adult
encodes a 38 kDa protein with 294 aminoacids. The SMN decade adulthood
protein is expressed in all somatic tissues and is highly
OMIM=Online Mendelian Inheritance in Man.
conserved from yeast to man.20–22 The SMN gene duplication
found in primates took place after the split of primates Table 1: Classification criteria for spinal muscular atrophy
from rodents, since mice have only one copy, which is
denoted Smn. SMN2, however, is unique to man because no exon seven (figure 3).25,26 The resultant SMNΔ7 protein
chimpanzees do not have this gene signature despite is non-functional and thought to be rapidly degraded.25,27,28
having multiple SMN gene copies in their genome.23 About 10% of SMN2 pre-mRNA is properly spliced and
More than 98% of patients with spinal muscular subsequently translated into full-length SMN protein.18
atrophy have a homozygous disruption of SMN1 by However, the efficiency of SMN2 splicing might be
deletion, rearrangement, or mutation.18,24 All these dependent on severity of disease, and production of a
patients, however, retain at least one copy of SMN2. This full-length transcript of SMN2 could range from 10%
gene undergoes alternative splicing and produces a to 50%.29 This low level of SMN protein allows embryonic
truncated mRNA isoform in which exon seven (major development, but is not sufficient to sustain the survival
product) or exon five, or both, are absent. A C-to-T of motor neurons of the spinal cord.
nucleotide transition at position six of exon seven (Ex7+6) In 2002, Cartegni and Kranier30 reported that the C-to-T
is responsible for the alternatively spliced isoform with nucleotide transition at Ex7+6 in SMN2 disrupts the

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Centromeric copy Telomeric copy

C272 C272
CATT–1 CMS1 CATT–1 C171 AG1–CA C212 C212 AG1–CA C171 CATT–1 CMS1
Cen Tel
GTF2H2 NAIP SMN2 SERF1 SERF1 SMN1 NAIP GTF2H2

SMA genetic map on chromosome 5q13

Figure 2: Genetic map of the spinal muscular atrophy locus

SMN gene copies are contained within two large inverted genomic fragments (black horizontal lines) within this region on chromosome 5q13. SMN1 (blue) is located
within the telomeric copy whereas at least one SMN2 (blue) is contained within the centromeric copy. Other genes in the vicinity of SMN copies are shown in black.
Blue and red arrows denote the direction of transcription of SMN and other genes, respectively. Multicopy microsatellite markers (red) are indicated within these
genomic repeats.

SR
SR
SF2 Tra2
U2 U2
ASF 100%
1
PA

SMN1 6 snRNP AF ESE 7 8 (A)n 6 7 8 (A)n

RN
hn

CAGACAA
~10%
SR
Tra2
~90%
RN 1
hn P A1

1
hn P A
PA

PA

SMN2 6 ESE 7 8 (A)n 6 8 (A)n

RN
RN

RN
hn

hn

U2 U2
snRNP AF
UAGACAA

Figure 3: Pre-mRNA splicing of SMN1 and SMN2

In SMN1, an exonic splicing enhancer (ESE), which contains the nucelotide cytosine (C) at position six in exon seven (Ex7+6), is recognised by splicing factor 2 or
alternative splicing factor (SF2/ASF), which interacts (thick black arrow) with the U2 class of small nuclear ribonuclear protein (U2 snRNP) to remove intron six. Other
splicing factors (eg, Tra2) determine splicing through interactions (thin black arrow) with ESE elements found centrally within exon seven. Serine and arginine
(SR)-rich proteins might also exert a positive splicing effect. In SMN2, the ribonucleotide uridine (transcribed from thymidine) at Ex7+6 favours exon seven exclusion
by binding to heterogeneous nuclear ribonuclear protein (hnRNP) A1, a negative splicing factor. Moreover, SF2/ASF no longer recognises this sequence motif.
Binding of hnRNP A1 is also believed to prohibit U2 snRNP binding to the branch point, which results in about 90% of SMN2 final mRNA transcripts with no exon
seven. The positive splicing factors downstream (thin black arrow) are functioning and could account for exon seven inclusion in about 10% of SMN2 transcripts.

function of an exonic splicing enhancer sequence, which
helps to promote normal splicing needed for production
of intact SMN protein. This putative exonic splice
enhancer sequence motif scored high for being dependent
on splicing factor 2 (SF2), a positive splicing factor that is
also known as the alternative splicing factor (ASF). The
SF2/ASF interacts with the U2 class of small nuclear
ribonuclear protein (U2 snRNP) and its auxiliary factor
(U2AF) at the branch point within intron six to help with
removal of the intron and successful pre-mRNA splicing
during SMN1 transcription (figure 3). Reconstruction of
this SF2/ASF-dependent exonic splice enhancer, by
reverting the thymidine on Ex7+6 in SMN2 gene to
cytosine, restored SMN2 splicing to include exon
seven.30
Kashima and Manley31 challenged the exonic splice
enhancer hypothesis, and argued that the C-to-T
nucleotide transition in exon seven creates an exonic
splice suppressor (ESS) in SMN2, which favours
exclusion of exon seven by binding heterogeneous
nuclear ribonuclear protein (hnRNP) A1 (a known
splicing repressor protein). Kashima and others32 showed
that C-to-T transition in SMN2 increases hnRNP A1
binding. The recruitment of hnRNP A1 might sterically
prevent the formation or stabilisation, or both, of the
snRNP complex needed for proper splicing. Disruption
of this hnRNP A1-dependent ESS with mutations or RNA
interference (RNAi) techniques restored SMN2 splicing
to include exon seven but had no effect on SMN1.31 This
restoration of exon seven inclusion in SMN2 is specific
to SMN.32 An A-to-G transition in intron seven (In7+100)
in SMN2 might create a second hnRNP A1 binding site
to assist further in the exclusion of exon seven.33 In an
effort to combine both theories, Cartegni and colleagues34
showed that hnRNP A1 might antagonise the SF2/ASF
exonic splice enhancer effect to promote exclusion of
exon seven, especially in SMN2, where this exonic splice
enhancer is inactivated. However, the exact mechanism

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surrounding exon seven inclusion or exclusion during
pre-mRNA splicing remains debatable.
SMN protein is expressed in the cytoplasm and nucleus
in all somatic tissues with particularly high amounts in
motor neurons of the spinal cord.35,36 Within the nucleus,
this protein localises to several small (0·1–2·0 μm
diameter), punctate structures associated with coiled
(Cajal) bodies named gems (gemini of coiled bodies).37,38
Although the exact cellular function of gems remains
unknown, cells from patients with spinal muscular
atrophy contain substantially fewer gems than do those
in gene carriers and controls without the disease.36,39
In eukaryotic cells, the spliceosome is the cellular
organelle that executes post-transcriptional pre-mRNA
splicing. Uridine-rich snRNPs (U snRNPs), the principal
components of spliceosomes, recognise highly conserved
sequences and ligate exons. SMN protein, part of a highly
stable complex with at least eight other proteins,40 is
necessary and sufficient for proper assembly of Smith
class core proteins in the U snRNP.41,42 A point mutation
(E134K) in the SMN tudor domain, the region that
mediates Smith class protein assembly,43 can cause the
disease by affecting the charge distribution in the
SMN-Smith class binding site.44 SnRNP assembly activity
is high during embryonic and postnatal development but
decreases after myogenic and neuronal differentiation.
Transcriptional networks involving many genes that
regulate cellular dynamics (eg, cell-cycle progression) are
highly active during this time.45,46 This RNA transcriptional
burden might need high amounts of snRNPs to
accomplish processing of RNA. Inhibition of snRNP
biogenesis by defective SMN protein might cause motor
neuron degeneration seen in patients with spinal
muscular atrophy. Winkler and co-workers47 noted that
impaired U snRNP biosynthesis in both frog (Xenopus
laevis) and zebrafish (Danio rerio) embryonic models
induced degeneration of motor neurons similar to that
seen in spinal muscular atrophy despite rather normal
development. Gabanella and others48 showed that
Smn–/–;SMN2+/+ mice have an eight-fold decrease in SMN
protein and a ten-fold decrease in snRNP assembly
activity in the brain and spinal cord compared with
normal (Smn+/+;SMN2+/+) mice.
Although SMN protein is expressed in all somatic cells,
why motor neurons of the spinal cord are specifically
vulnerable in spinal muscular atrophy is puzzling.
Studies suggest ´ that SMN protein might have an
important part in certain cellular functions that are
unique to motor neurons. Investigators noted that this
protein was localised in ribonucleoprotein granules in
the neurites and growth cones of primary motor neurons,
and in motor neurons derived from embryonic stem
cells.49,50 Live-cell fluorescence microscopy was used to
observe active bidirectional transport of these granules to
neuronal processes and growth cones.50 SMN protein
also interacts with hnRNP R and Q.51,52 hnRNP R interacts
with the 3´ untranslated region of β-actin mRNA.51 Low
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Seminar
concentration of SMN protein resulted in low titres of
β-actin mRNA and protein in axons and growth cones,53
suggesting that SMN protein might be involved in
transportation of ribonucleoprotein complexes containing
β-actin. Deficiency of β-actin results in axonal outgrowth
and pathfinding defects in two neuronal culture models54
and in a zebrafish model of spinal muscular atrophy.55
Profilin, an actin-binding protein, regulates actin
dynamics through sequestration and release of actin
monomers. SMN protein colocalises with profilin IIa
(the predominant isoform in neuronal tissue56) as distinct
granules in neurite-like extensions and growth cones.57
Mutant SMN protein fails to interact with profilin IIa,
and knocking down profilin IIa alone inhibits neurite
outgrowth.57 Setola and colleagues58 reported the
identification of a novel SMN isoform named axonal-SMN
that is preferentially transcribed from SMN1 and includes
intron three. The repetitive polyadenine tract of this
intron was unexpectedly resistant to mutations, which
would alter the C-terminal protein structure, suggesting
that this sequence might be needed for the structure or
function, or both, of axonal-SMN.59 The axonal-SMN
protein was not detected in the glia or grey matter outside
Rexed’s lamina IX (motor neuron area) in the spinal
cord.58 Several groups have made disease models in
Drosophila melanogaster that show neuronal and muscular
defects.60,61 These models have led to postulation of roles
for SMN protein at the neuromuscular junction61 and in
skeletal muscle.60 These data suggest that SMN protein
might sustain the survival of motor neurons by allowing
normal axonal transport and maintaining the integrity of
neuromuscular junctions. Low concentrations of SMN
protein might be specifically detrimental to motor
neurons because of the long axons and their unique
interactions with skeletal muscles.
The mouse Smn gene, located on chromosome 13,
contains nine exons like the human SMN gene and is
83% homologous to the SMN1 open reading frame.62,63
No alternative splicing isoform exists since only a
full-length 1·4 kb transcript has been noted. Creating a
mouse with spinal muscular atrophy was challenging
because homozygous deletion of murine Smn (Smn–/–) is
lethal at the embryonic stage,20 whereas heterozygous
Smn+/– mice are phenotypically normal. These early
failures suggested that more innovative approaches were
needed to establish a mouse model of the disease.
In the past 7 years, several different models of spinal
muscular atrophy have been developed in mice.64
Taiwanese scientists used the observation that patients
with this disease always have at least one copy of SMN2
and created a transgenic (Tg) mouse with human
SMN2.65 Smn+/– mice were crossed with Smn+/–;TgSMN2
mice to produce Smn–/–;TgSMN2, with clinically and
histopathologically similar spinal muscular atrophy to
that in people. Similar strategies were used by other
groups to create model mice that mimicked human
spinal muscular atrophy with varying severities in
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clinical symptoms and histopathology.66–68 Smn–/–;TgSMN2
produced in different laboratories64,67 have varying
phenotypes, presumably because of different genetic
backgrounds, different allelic knockouts, or different
sizes of transgene regions. By purifying the genetic
background to standardise the number of SMN2 copies,
a mouse with type III spinal muscular atrophy with no
phenotypic heterogeneity was created.69 Tissue-specific
knockdown of SMN protein has also been accomplished
in neurons70 or muscle,71,72 by flanking Smn with loxP
sequences (floxed) and expressing Cre recombinase
under a neuronal-specific or muscle-specific promoter.
Expression of Cre recombinase results in efficient
deletion of floxed genes by homologous recombination.
Constitutive overexpresssion of SMN protein has also
been accomplished in neuronal and skeletal muscle
tissue.73 Gavrilina and others73 showed that neuronal
overexpression significantly prolonged Smn–/–;SMN2+/+
mice, but overexpression in skeletal muscle had no
effect. Butchback and colleagues74 created an
Smn–/–;SMN2+/+;SMNΔ7+/+ mouse whose phenotype is
slightly less severe (lifespan 14–15 days).64,67
Clinical implications of cloning SMN genes
Identification of the SMN genes not only enabled further
research into the molecular pathogenesis of spinal
muscular atrophy, but also allowed rapid and sensitive
diagnosis. Figure 4 shows the algorithm for diagnosis of
the disease by molecular genetic methods. Patients with
a clinical presentation resembling spinal muscular
atrophy should be tested for homozygous deletion of the
SMN1 gene, which is 95% sensitive and about 100%
specific.75,76 Two diagnostic tests exist: a combined PCR
with restriction enzyme digestion assay77,30 and an allele-
specific PCR assay of the SMN1 and SMN2 PCR
products.78,79 The first method uses differential sus-
ceptibility of the SMN1 and SMN2 PCR products to
endonuclease digestion. The allele-specific PCR assay

SMA with typical or

atypical clincial features

SMN1 gene deletion test Homozygous SMN1 5q SMA diagnosis

deletion detected confirmed

Homozygous SMN1
deletion not detected

Repeat clinical examination,

EMG, NCS, and CK

Demyelinating or axonal Atypical SMA features, Diffuse weakness, Proximal > distal weakness,
neuropathy, NMJ disorder, neurogenic electromyography, normal electromyography, neurogenic electromyography,
myopathy, muscular dystrophy normal CK normal CK, normal NCS normal CK

Consider muscle or nerve biopsy; Consider other motor neuron Consider brain or spinal cord SMN1 gene copy count One SMN1 copy
genetic tests for myopathies, disorders (SMARD, X-linked MRI; metabolic screens
muscular dystrophies, and SMA, distal SMA, ALS)
neuropathies

Two SMN1 copies SMN1 gene sequencing SMN1 mutation found

No SMN1 mutation found 5q SMA diagnosis

confirmed

SMN-related SMA
diagnosis remains
unconfirmed

Figure 4: Diagnostic algorithm for spinal muscular atrophy

Any patient presenting with clinical symptoms resembling spinal muscular atrophy (SMA) should be tested for homozygous deletion of SMN1, which would confirm the diagnosis of SMN-associated
SMA (5q SMA). A negative SMN1 test should be followed with a repeat clinical examination for atypical clinical features (eg, contractures, eventration of hemidiaphragms, congenital absence of
muscles, pes equines deformity) and laboratory testing for creatinine phosphokinase (CK) and electrophysiological studies such as electromyography (EMG) and a nerve conduction study (NCS). If
lower motor neuron disease is suggested by EMG, SMN1 copy number will establish if SMN1 sequencing is indicated to identify intragenic mutations in patients with a single SMN1 copy. When two
SMN1 copies are detected, then investigation should be directed towards other motor neuron diseases by further diagnostic work-up such as muscle or nerve biopsy, imaging studies, metabolic
screens, and genetic testing. ALS=amyotrophic lateral sclerosis. NMJ=neuromuscular junction. SMARD=SMA with respiratory distress. Red boxes=diagnostic assessments. Blue boxes=physical and
laboratory findings. Green boxes=final diagnoses.

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uses the nucleotide variation in exon seven and
specifically designed PCR primers to quantitatively
amplify SMN1 or SMN2. Both tests confirm a diagnosis
of 5q spinal muscular atrophy, but the second one
provides a quantitative measure of SMN1 copy number.
In cases in which a single copy of SMN1 is detected,
further investigation into SMN1 intragenic mutations by
gene sequencing is warranted. When two SMN1 copies
are detected, further diagnostic studies should investigate
other motor neuron, brain, spinal cord, and
neuromuscular junction diseases (figure 4).
The phenotypic heterogeneity of spinal muscular
atrophy is striking considering that nearly all patients
have a defect in the same SMN1 gene. After initial
hypotheses that the disease phenotype correlates with
the size of the genomic deletion,80 a thorough search of
this unstable genetic region revealed multiple SMN2
copies in the human genome,81,82 possibly caused by gene
duplication or conversion events, or both.16,83 Investigation
with Smn–/– mice showed that adding a human SMN2
transgene could rescue pups from early embryonic
death,20 and eight copies of human SMN2 could produce
a phenotypically normal mouse.67 Other studies in man
also showed that a higher number of SMN2 copies
correlates with milder phenotypes.81 However, variations
in phenotypes are common with any number of SMN2
copies. Therefore, prediction of the spinal muscular
atrophy phenotype with SMN2 copy number is not
recommended. Some evidence suggests that other
phenotype-modifier genes—eg, GTF2H2 (BTF2P44),
SERF1A (H4F5)—might exist.84,85 Evidence also suggests
that higher amount of SMN protein is observed in
patients with milder form of spinal muscular atrophy.86
High homology between SMN1 and SMN2 complicated
carrier detection with PCR methods, because
amplification of an SMN1 fragment frequently amplifies
SMN2 as well. Initial carrier detection methods used
high sensitivity densitometry of PCR products to identify
copy number.82 Present methods use quantitative
real-time PCR allowing measurement of both SMN1 and
SMN2 copy number separately.78,80,87,88 However, the SMN1
copy number test might rarely and inaccurately determine
the carrier status, because about 3·2% of the general
population has both SMN1 copies on one chromosome
and none on the other (denoted 2+0).89,90 These people are
carriers because one of their chromosomes is missing a
normal SMN1 allele. Another 1·7% have intragenic
mutations that are not detectable by these methods of
SMN1 dosage analysis.80 SMN1 gene sequencing can
detect intragenic mutations, and 2+0 carriers can be
detected by karyotyping with fluorescence in-situ
hybridisation.
Chorionic villi sampling and amniocentesis can
effectively find out the fetal genotype.91,92 Additionally,
circulating fetal cells in maternal blood can be isolated by
isolation by size of epithelial tumour or trophoblastic
cells (ISET) for PCR-based genetic analysis.93 For those
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who do not want to wait for the decision until early
pregnancy, in-vitro fertilisation allows several blastomeres
to be tested for SMN1 deletion after growth in culture.
After the allele-specific PCR assay of a single cell is done,
unaffected embryos can be implanted.94–96 This procedure
requires high technical precision and is available only at
select centres.
Treatments to prevent motor neuron death will
presumably have the greatest effect before onset of disease.
The challenge, however, is the identification of these
patients before they are symptomatic. Carrier parents are
asymptomatic, and most children with spinal muscular
atrophy appear normal for the first few months of life.
Newborn screening could address this challenge, but it has
not become standard practice because without effective
treatment this practice is not justified. A study assessed
the effectiveness of genetic screening for SMN1 in neonates
with real-time quantitative PCR on 153 dried blood spots
on filter paper.97 With 100% specificity and sensitivity, this
study provided evidence of the feasibility for large-scale
newborn screening. However, social, ethical, and political
implications, as well as implementation of procedures and
treatment protocols, will need to be sufficiently addressed
before screening of newborn babies can be advocated.
Treatment
One substantial implication of uncovering the molecular
genetic basis of spinal muscular atrophy is in the devel-
opment of candidate therapeutics. The unique genomic
organisation of the SMN genes allows a novel therapeutic
approach: to promote SMN2, existing in all patients, to
function like the missing SMN1 gene. This idea prompted
investigations into increasing inclusion of exon seven in
SMN2 mRNA transcripts,39,98–101 upregulation of SMN2
transcription by promoter activation,102–104 modulation of
SMN protein translation,105–107 and prevention of SMN
protein degradation.28,108
Serine–arginine-rich proteins are splicing factors that
interact with exonic splice enhancers and affect mRNA
splicing. Htra2-β1, a serine–arginine-rich-like splicing
factor, was found to stimulate full-length SMN2
expression109 by interacting with exonic splice enhancers,
especially elements that contain AG-rich motifs such as
positive splicing enhancer 2.26 Abrogation of such motifs
results in skipping of exon seven.26 Little is known about
the importance of Htra2-β1, and SMN genes are the only
known human genes whose splicing is specifically
regulated by Htra2-β1; other human isoforms (eg,
Htra2-β3 and Htra2-α) did not stimulate proper SMN2
splicing.109 The use of pharmacological compounds to
upregulate Htra2-β1 might prove to be a valuable strategy
to modulate SMN2 splicing.
One of the first candidate treatments for spinal
muscular atrophy was aclarubicin (aclacinomycin A), an
anthracycline and topoisomerase II inhibitor used in
patients with solid tumours or leukaemia. Aclarubicin
was discovered by screening a 960-member combinatorial
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chemistry library with a semiquantitative assay based on
RT-PCR to detect inclusion of exon seven.99 Aclarubicin’s
toxicity prohibited further development for clinical use to
treat patients with spinal muscular atrophy. Nevertheless,
it served as a proof of principle for identifying modifiers
of SMN2 splicing and that high throughput screening
was a valid approach to identify candidates.
With histone acetylation, the surrounding DNA relaxes
and is accessible to transcriptional machinery. Inhibition
of histone deacetylation might enhance expression in
about 2% of human genes.110 Sodium butyrate was the
first histone deacetylase inhibitor found to enhance
SMN2 expression and increase exon seven inclusion in
lymphoid cell lines derived from patients with spinal
muscular atrophy. This treatment also increased
expression of SMN protein in the spinal cord and motor
neurons of SMA-like mice developed by the Taiwanese
researchers described previously.99 Not surprisingly, the
expression of other genes (eg, globin) was also increased.
However, the short half-life, about 6 min in human
serum, prohibited sodium butyrate from substantial
clinical development. Encouraged by the effect of sodium
butyrate, the search began for other putative histone
deacetylase inhibitors. In 2003, two groups reported that
valproic acid, a histone deacetylase inhibitor approved for
epilepsy treatment, increased full-length SMN mRNA.
Valproic acid activated the SMN2 promoter and induced
expression of positive splicing factors, SF2/ASF and
Htra2-β1.100,101 In 2004, Andreassi and colleagues103
reported that a sodium butyrate derivative, phenylbutyrate,
increased full-length transcript production from SMN2,
SMN protein, and the number of nuclear gems.
Phenylbutyrate is still used to treat children with urea-
cycle defects and has a slightly longer half-life (0·8–1·0 h
in human serum) than has sodium butyrate.111
Further investigations were directed towards more
potent and specific histone deacetylase inhibitors.
Suberoylanilide hydroxamic acid, a potent histone
deacetylase inhibitor under clinical investigation for
cancer therapy,112 has good oral bioavailability and is
non-toxic, despite complete inhibition.113 At low
micromolar concentration, suberoylanilide hydroxamic
acid activated the SMN2 gene in fibroblasts from patients
and increased SMN protein in rat motor neuron cultures,
and in both human and rat hippocampal brain slice
cultures.113
Another potent hydroxamic acid, trichostatin A, was
originally isolated in 1976 as an antifungal drug,114 but it
has not been in clinical use because of the high cost of
isolation and purification. Treatment with trichostatin A
in normal and SMNΔ7 mouse models specifically
increased properly spliced SMN2 transcript, snRNP
assembly, and SMN protein in neural and muscle tissue.115
Even after the onset of disease, trichostatin A treatment
was able to extend survival and increase the size of
myofibres and spinal cord motor neurons in the SMNΔ7
mouse model. Jarecki and colleagues104 screened
2126
550 000 compounds in a β-lactamase assay designed to
detect activation of the SMN2 promoter. They discovered
five classes of compounds that altered SMN2 transcript.
Four of these increased the full-length to truncated SMN2
transcript ratio. Two also increased SMN protein and the
number of nuclear gems in fibroblasts from type I
patients. Further medicinal chemistry and structure-
activity relationship studies will assess the promise of
these molecules in clinical use.
Grzeschik and colleagues39 noted that hydroxyurea, a
ribonucleotide reductase inhibitor, increased the full length
to truncated SMN mRNA ratio. The total amount of SMN
mRNA in the lymphoblastoid cell lines from patients with
spinal muscular atrophy was not changed after hydroxyurea
treatment, but the increase in full-length SMN mRNA was
coupled by a decrease in truncated SMN mRNA. This
finding shows that hydroxyurea increased the full-length
SMN mRNA by promoting exon seven inclusion rather
than activating the SMN2 promoter.39 Furthermore,
hydroxyurea treatment, in vitro, increased SMN protein
and the number of nuclear gems. Hydroxyurea also has a
good safety profile, easy oral administration, and high
bioavailability. Riluzole is a neuroprotective agent known
to promote neuronal survival through glutaminergic
antagonism.116 Haddad and colleagues117 treated special
spinal muscular atrophy mice with Smn exon seven deleted
only in neurons and found that riluzole extended lifespan
but did not significantly improve function. It prevented
aberrant cytoskeletal organisation at the motor terminals
but did not prevent loss of proximal axons.117 After screening
about 47 000 compounds, Lunn and others107 discovered
that indoprofen, a non-specific cyclo-oxygenase inhibitor
and a non-steroidal anti-inflammatory drug, increased
SMN protein in fibroblasts of patients with type I spinal
muscular atrophy. It also increased the number of nuclear
gems and caused a trend towards increased viability in the
Smn–/–;TgSMN2 spinal muscular atrophy mouse model.107
No other non-steroidal anti-inflammatory drugs that were
tested showed a similar effect, suggesting that indoprofen’s
effect is independent of cyclo-oxygenase inhibition. A
previously approved drug in the UK, indoprofen, was
removed from the market because of high frequency of
gastrointestinal bleeding secondary to cyclo-oxygenase
inhibition. Other compounds—eg, a β-adrenergic
agonist,118 an Na+/H+ exchanger inhibitor,119 and polyphenol
plant compounds120—have been reported to promote exon
seven inclusion and to increase SMN protein concentration
in fibroblasts derived from patients with spinal muscular
atrophy.
Translation of SMN1 mRNA ends at a stop codon at the
end of exon seven with no translation of exon eight. The
16 aminoacids absent in SMNΔ7 protein, although not a
direct mediator of SMN’s numerous activities, are
believed to be required for oligomerisation of SMN
protein.121,122 Heterologous sequences are able to
substitute123 for the cytoplasmic localisation signal of
SMN protein.50 Since aminoglycosides have been reported
www.thelancet.com Vol 371 June 21, 2008
 Seminar

to promote stop codon read-through,124 the Lorson
group105,106 reported that various aminoglycosides in-
creased the number of nuclear gems and full-length
SMN protein by preventing recognition of stop codon in
the SMN transcript; this read-through results in the
addition of a non-specific tail needed for proper cyto-
plasmic localisation of the SMN gene and increased
activity of SMNΔ7 protein.
Another way to modulate SMN2 splicing was to use
exon-specific antisense oligonucleotides that were
complementary to regions of SMN2 exon seven.125,126
Antisense oligonucleotides bound to pre-mRNA
inhibited binding of negative splicing factors, which led
to increased exon seven inclusion, the amount of SMN
protein, and the number of nuclear gems in fibroblasts
from patients with spinal muscular atrophy. Some
bifunctional antisense oligonucleotides also have a
non-complementary tail that is rich in potent exonic
splice enhancer sequences to recruit trans-acting
positive factors that might inhibit binding of negative
splicing proteins.126 Antisense oligonucleotides were
able to modulate splicing and inhibit signalling in a
mouse study.127 Further investigations into bifunctional
antisense oligonucleotides might increase potency but
run the risk of being incompatible as a treatment option
because of possible endogenous immunological

Design SMA type Sample size Primary outcome Secondary outcome Results Comments
measure measure
Acetyl-L-carnitine MC, randomisation, DB, II, III 110 Handgrip and elbow Knee flexion strength, Completed, results pending http://www.enmc.org/
PC flexion strength forced vital capacity, workshop/?id=73
quality of life
Albuterol131 SC, OL, PS II, III 13 (5 type II, Muscle strength, .. Significant increase in all No placebo control,
8 type III) forced vital capacity, outcome measures unblinded. Improvement
lean body mass could be due to normal
growth
Creatine MC, randomisation, PC II, III 55 GMFM Functional tests, Completed, results pending No effect
pulmonary function tests
Gabapentin136,140 MC, randomisation, DB, II, III 84 (40 treatment, Muscle strength Forced vital capacity, No difference between ..
PC 44 placebo) testing SMA rating scale, mini- treatment and placebo
sickness impact profile groups
MC, randomisation, II, III 120 (61 treatment, Change in maximum Forced vital capacity, Improvement in leg strength ..
treatment vs non 59 no treatment) voluntary isometric timed tests and a trend for improvement
treatment contraction in arm strength
Hydroxyurea141 SC, PI or PII, I 18 (2:1 treated to Survival MUNE, SMN mRNA and In progress ClinicalTrials.gov
randomisation, DB, PC placebo ratio) protein Identifier: NCT00083746
SC, PI or PII, II or III 24 (2:1 treated to GMFM MUNE, SMN mRNA and In progress ClinicalTrials.gov
randomisation, DB, PC placebo ratio) protein Identifier: NCT00084006
SC, PII, randomisation, II or III 60 SMN mRNA and .. In progress ClinicalTrials.gov
DB, PC protein, motor Identifier: NCT00485511
function, lung
function
SC, OL, PS II, III 33 HFMS SMN mRNA Completed ..
Phenylbutyrate134–136 MC, PI or PII, OL, PS II 29 (10 treatment, Tolerability HFMS, myometry, forced Significant increase in ..
19 no treatment) vital capacity HFMS scores
SC, OL, PS II, III 6 (4 type II, SMN mRNA level .. Increased SMN mRNA level ..
2 type III) and 3
carriers
MC, PII, randomisation, II 107 (54 treatment, HFMS Myometry, forced vital No significant improvement ..
DB, PC 53 placebo) capacity
MC, PI, II, III 30 Tolerability SMN mRNA and protein In progress ClinicalTrials.gov
OL Identifier: NCT00439569
MC, PI, I 30 Tolerability Pharmacokinetics, SMN In progress ClinicalTrials.gov
OL mRNA and protein Identifier: NCT00439218
SC, PI or PII, OL II, III 12 Safety, tolerability Overall motor function In progress ClinicalTrials.gov
Identifier: NCT00528268
SC, OL, PS I, II 20 Safety SMN mRNA and protein, Completed, results pending ··
TIMP, HFMS
Riluzole139 MC, PI, randomisation, I 10 (7 treatment, Survival .. No adverse event or serious Trial prematurely stopped
DB, PC 3 placebo) adverse event attributed to due to withdrawn
treatment financial support
MC, PI or II, OL I 40 (targeted), Survival SMN mRNA Completed, results pending ..
3 (completed)
(Continues on next page)

www.thelancet.com Vol 371 June 21, 2008 2127

 Seminar

Design SMA type Sample size Primary outcome Secondary outcome Results Comments
measure measure
(Continued from previous page)
Somatotropin MC, PII, randomisation, II, III 20 Hand-held myometry Functional tests, Not yet recruiting ClinicalTrials.gov
DB, PC, crossover pulmonary function Identifier: NCT00533221
testing
Thyrotropin- SC, randomisation, DB, II, III 9 (6 treatment, Muscle strength using NCV, CMAP Improvement muscle Patient served as own
releasing hormone PC 3 placebo) hand-held strength and some CMAP/ control because of small
(protirelin)142 dynamometry NCV values sample size
Valproic acid137,138 SC, OL, PS I, II, III 21 (5 type I, SMN2 mRNA level .. SMN2 mRNA increased in ..
10 type II, 6 type III) 7 patients with SMA,
and 10 carriers unchanged or decreased in 13
SC, Retrospective, OL III, IV 7 Hand-held .. Strength improvement by ..
dynamometry 48%
SC, PII, randomisation, Type III 36 Muscle strength MUNE, lean body mass, In progress ClinicalTrials.gov
DB, PC, crossover ambulatory timed tests Identifier: NCT00481013
SC, PI or PII, OL, PS I, II, III 42 Tolerability SMN mRNA and protein, Completed ClinicalTrials.gov
(>2 years) EMG, pulmonary Identifier: NCT00374075
function testing
Valproic acid and PII, randomisation, DB, Cohort 1: 90 (in two cohorts) Safety, HFMS, SMN mRNA, MUNE, In progress ClinicalTrials.gov
L-carnitine PC, crossover sitting II and myometry CMAP, pulmonary Identifier: NCT00227266
III; cohort 2: function testing
standing II
and III

The information contained in the ongoing studies was gathered through unpublished sources, and some might not be current or complete. CMAP=compound motor unit action potentials. DB=double-blind.
GMFM=gross motor function measure. HFMS=Hammersmith functional motor score. MC=multicentre. MUNE=motor unit number estimation. NCV=nerve conduction velocities. OL=open-label.
PC=placebo-controlled. PI=phase I. PII=phase II. PIII=phase III. PS=pilot study. SC=single centre. TIMP=test of infant motor performance.

Table 2: Completed and ongoing clinical trials of various therapeutic agents for spinal muscular atrophy

reactions. Cartegni and Krainer128 developed a peptide
nucleic acid oligonucleotide mimetic that is resistant to
nucleases and does not activate ribonuclease H on
binding to the target RNA. High amounts of this nucleic
acid mimetic increased exon seven inclusion in a
three-exon (exon six through eight) mini-gene splicing
reaction in vitro. This effect was sequence specific,
because a peptide nucleic acid directed against another
gene had no effect. DiMatteo and colleagues129 used
single-stranded oligonucleotides to convert SMN2 into
SMN1 in fibroblasts of patients with spinal muscular
atrophy. The conversion was confirmed by quantitative
RT-PCR and by proper intracellular SMN protein
localisation.
Clinical trials
Many compounds noted to increase SMN mRNA and
protein expression in vitro and in animal models in the
past few years led to human studies of candidate
therapeutics.130 Small-scale studies in man with candidate
compounds that have no supporting preclinical data have
met little success.131,132 Several clinical trials based on
strong pre-clinical data are in progress. Early pilot studies
of phenylbutyrate in patients with types II and III spinal
muscular atrophy showed increases in full-length SMN
expression and in motor function as measured by
myometry and the Hammersmith functional motor
scale.133,134 A randomised, double-blind, placebo-controlled
trial of phenylbutyrate in 107 patients with type II disease
at ten Italian centres, however, reported no significant
change in functional motor scores after 13 weeks of
treatment.135 Similarly, a placebo-controlled, randomised,
double-blind trial of gabapentin in types II and III
patients showed no substantial difference in muscle
strength between treatment and placebo groups after
12 months of treatment.136 Treatment of patients with
spinal muscular atrophy and disease-gene carriers with
valproic acid in a pilot study resulted in an increase in
full-length SMN expression, but no clinical outcome was
measured.137 An open-label study of valproic acid in seven
patients with mild disease (types III and IV) showed a
rise in quantitative muscle strength by dynamometry
and improved subjective motor functioning after a mean
treatment duration of 8 months.138 Riluzole was tested in
a phase I, randomised, double-blind, placebo-controlled
study for patients with type I disease.139 Although this
study lacked statistical power because of a small sample
size, benefit was suggested in treated patients. A
multicentre randomised trial intended to further study
the effect of riluzole in type I has been completed;
however, the result is pending. Several randomised trials
are in progress, including a multicentre trial with
combined l-carnitine and valproic acid; two double-blind,
placebo-controlled trials with hydroxyurea in type I and
in types II or III patients; and a multicentre trial
assessing the efficacy of somatotropin in type II and III
patients. Table 2 lists the clinical trials completed and
in progress.

2128 www.thelancet.com Vol 371 June 21, 2008


Conclusion
Future advances in high-throughput screening technol-
ogies, computation, and organic synthesis143 will allow
development of specific, non-toxic, potent small molecules
to modulate splicing and expression of SMN2.
Government-backed programmes, such as the SMA
Project, will accelerate these goals. Additionally, modulation
of non-SMN targets (eg, Gemin2 and Gemin6) can affect
SMN protein activity.144 Promising, alternative gene therapy
approaches to modulate pathogenesis with viral vectors to
deliver SMN1 directly,145 trans-splicing RNAs to correct
SMN2 splicing,146 and neurotrophic agents (ie,
cardiotrophin-1)147 should be explored. Motor neurons
derived from stem cells have been established,148,149 and
stem cells are promising for use in assays and human
therapeutic trials. Neuronal stem cells proved to protect
surviving motor neurons in rats with motor neuron
injury.150 In an exciting report, embryonic stem cells derived
from motor neurons survived in spinal cords of rats with
motor neuron injury, and axons from these cells were
Panel: Databases and websites
Databases
• Online Mendelian Inheritance in Man (NCBI/OMIM):
http://www.ncbi.nlm.nih.gov/omim/
• PubMed: http://www.pubmed.com
Clinical trial networks in the USA
• American SMA Randomised Trial (AmSMART) Group:
http://www.amsmart.org
• Pediatric Neuromuscular Clinical Research Network
(PNCR): http://www.urmc.edu/sma
• Project Cure SMA: http://www.projectcuresma.org
Clinical trial networks in Europe
• European Neuromuscular Centre (ENMC):
http://www.enmc.org
• TREAT-NMD: http://www.treat-nmd.eu
Clinical trial registry in the USA
• ClinicalTrials.gov: http://www.clinicaltrials.gov
Patient and family advocacy groups
• The Benjamin Foundation:
http://www.thebenjaminfoundation.org
• Families of SMA: http://www.fsma.org
• Fight SMA/Andrew’s Buddies: http://www.fightsma.org
• Hope and Light Foundation: http://www.hopeandlight.org
• Miracle for Madison and Friends:
http://www.miracleformadison.org
• Payton’s Pals: http://www.paytonspals.com
• SMA Angels Charity: http://www.smaangels.org
• SMA Foundation: http://www.smafoundation.org
• SMA Support: http://www.smasupport.com
Patient registry
• International SMA Patient Registry: http://www.iupui.
edu/~medgen/hereditary/sma.html
www.thelancet.com Vol 371 June 21, 2008
Seminar
noted in ventral roots.151 Moreover, these motor neurons
formed neuromuscular junctions and induced contraction
in vitro with cultured myoblasts. Future research would
need to aim at delivering these stem cells derived from
motor neurons to the spinal cord, countering host versus
graft immunological responses, and guiding axonal For more on the SMA project
outgrowth to reach the target muscles. see http://www.smaproject.org
As clinical trials have progressed, several challenges
have surfaced.151 The small number of patients for clinical
trials will need a coordinated effort for patients to
participate in different trials. Criteria will need to be set up
for the selection of compounds for clinical trials. Various
elements of trial design will need to be unified to allow
comparisons of results between trials. Sensitive and valid
outcome measures will need to be developed.130 Few
clinical outcome measures are available for patients with
type I spinal muscular atrophy other than survival.
Although myometry using hand-held dynamometers152
and quantitative muscle testing153,154 are reliable muscle
strength measures in types II and III patients, these are
not useful for type I patients. Functional motor scales,
such as the Hammersmith functional motor scale and
gross motor functional measurement, are also restricted
to types II and III patients.155 Measuring the function of
other organ systems, such as pulmonary function and
ability to swallow and speak, might be sensitive outcome
measures, especially in younger patient groups.
Additionally, biomarkers, such as SMN mRNA and protein
and gem count, might be sensitive outcome measures for
clinical trials. The amounts of full-length SMN transcripts
and SMN protein are well correlated with disease severity.
As such, several groups have developed promising assays
to monitor changes in SMN mRNA and protein in human
blood.156,157SMN mRNA seems to be the most amenable to
large multicentre trials.158
The clinical implications of elucidating the molecular
genetic basis of spinal muscular atrophy and advances in
medical technology have changed the clinical care of
patients. However, these patients still receive highly
diverse care for several reasons such as large clinical
phenotypic variation, multiorgan system involvement,
geographical variation in availability of medical expertise,
different values of clinicians and families, and variations
in financial resources. Differences in clinical care also
hinder valid measures of clinical outcomes in clinical
trials. The International Standard of Care Committee for
spinal muscular atrophy was established in 2005 with a
mission to set a standard of care. The committee consists
of 12 core members and four working groups: diagnostics
or new interventions, pulmonary, gastroenterology or
nutrition, and orthopaedics or rehabilitation. Each
working group contains eight to ten experts in that
specialty. The groups identified few data that were
available to establish evidence-based practice guidelines
for the disease. Hence, a Delphi survey was used to
achieve consensus among more than 60 experts from
12 countries. The committee held two meetings to draft a
2129
 Seminar
consensus statement for the standard of care for patients.7
As a result, we hope that receiving consistent medical
care will become easier for patients and that valid clinical
outcomes can be measured during clinical trials.
Although major challenges remain towards developing
a therapeutic agent for patients with spinal muscular
atrophy, our understanding of the disease biology,
including several animal models, has grown exponentially
since identification of SMN1 in 1995. Clinical trials have
begun and have uncovered areas to improve the design
of human studies and patient care (panel). Further
research and development, coupled with a broadly
accepted and applied standard of care for these patients,
will help us manage, treat, and eventually cure this
devastating neurodegenerative disease.
Conflict of interest statement
We declare that we have no conflict of interest. We have no relation to
any patent application or intellectual property for any spinal muscular
atrophy therapeutic candidate.
Acknowledgments
We thank Rishi Bhatnagar for medical illustration and Hannes Vogel for
providing histopathological slides. MRL is supported by the Molecular
Basis of Medicine scholarly concentration at Stanford University School
of Medicine.
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