Clinical Therapeutics/Volume 36, Number 1, 2014
Molecular Therapeutic Strategies for Spinal Muscular
Atrophies: Current and Future Clinical Trials
Chiara Zanetta, MD; Monica Nizzardo, PhD; Chiara Simone, PhD; Erika Monguzzi, PhD;
Nereo Bresolin, MD; Giacomo P. Comi, MD; and Stefania Corti, MD, PhD
Dino Ferrari Centre, Neuroscience Section, Department of Pathophysiology and Transplantation (DEPT),
University of Milan, Neurology Unit, IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico,
Milan, Italy
ABSTRACT
Background: Spinal muscular atrophy (SMA) is an
autosomal recessive motor neuron disease caused by
mutations in the survival motor neuron gene (SMN1)
and the leading genetic cause of infant mortality.
Currently, there is no effective treatment other than
supportive care.
Objective: This article provides a general overview
of the main aspects that need to be taken into account
to design a more efficient clinical trial and to summa-
rize the most promising molecular trials that are
currently in development or are being planned for
the treatment of SMA.
Methods: A systematic review of the literature was
performed, identifying key clinical trials involving
novel molecular therapies in SMA. In addition, ab-
stracts presented at the meetings of the Families of
Spinal Muscular Atrophy were searched and the
Families of Spinal Muscular Atrophy Web site was
carefully analyzed. Finally, a selection of SMA clinical
trials registered at clinical- trials.gov has been included
in the article.
Results: The past decade has seen a marked
advancement in the understanding of both SMA
genetics and molecular mechanisms. New molecules
targeting SMN have shown promise in preclinical
studies, and various clinical trials have started to test
the drugs that were discovered through basic research.
Conclusions: Both preclinical and early clinical trial
results involving novel molecular therapies suggest
that the clinical care paradigm in SMA will soon
change. (Clin Ther. 2014;36:128–140) & 2014 Elsev-
ier HS Journals, Inc. All rights reserved.
Key words: clinical trials, gene therapy, ISIS-
SMNRx, olesoxime, oligonucleotides, small molecules,
spinal muscular atrophy.
128
INTRODUCTION
Spinal muscular atrophies (SMAs) are a group of
hereditary autosomal recessive neuromuscular dis-
eases that are characterized by the degeneration of
motor neurons in the spinal cord and brainstem,
resulting in progressive proximal muscle weakness,
hyposthenia, and paralysis, which are usually sym-
metrical. SMA is the most frequent genetic cause of
infant mortality, with an estimated incidence of 1 in
6000 to 1 in 10,000 live births and a carrier frequency
of 1 in 40 to 1 in 60.1,2 The classical form of the
disorder is caused by a genetic mutation3 in the
5q11.2-q13.3 locus, which affects the survival motor
neuron (SMN) gene4 and leads to the reduction of
SMN protein. SMA is clinically conventionally
classified into 4 phenotypes (I, II, III, and IV) on the
basis of age of onset and highest motor function
achieved, with an additional phenotype (type 0) to
describe the severe forms with an antenatal onset.5
Prognosis depends on the phenotypic severity, ranging
from high mortality within the first year for SMA type
I to no mortality for the chronic and later-onset forms.
The increased attention to early diagnosis and to several
aspects of management of SMA has stimulated the
development of clinical guidelines and standards of
care.6,7 In the past decade, many promising new
therapeutic approaches have been tested in clinical trials
of patients with SMA8–11 but with limited or no success.
At the present, no effective therapy is available for SMA,
besides supportive care. Consequently, the development
of novel therapies in SMA now has strong academic,
government, and industry involvement, in addition to
Accepted for publication November 22, 2013.
http://dx.doi.org/10.1016/j.clinthera.2013.11.006
0149-2918/$ - see front matter
& 2014 Elsevier HS Journals, Inc. All rights reserved.
Volume 36 Number 1
 C. Zanetta et al.

the interest of several parental organizations and foun-
dations. The goal of the present article was to summa-
rize the literature on the emerging molecular therapeutic
approaches that are currently being investigated or
planned to be tested in clinical trials of SMA (Figure 1).
METHODS
A systematic review of the English-language articles
listed in PubMed over the past 10 years was per-
formed by using the following key words: spinal
muscular atrophy, SMA, oligonucleotides, gene ther-
apy, molecular therapy, and small molecules. All
articles found were systematically analyzed and taken
into consideration when preparing the review. In
addition, abstracts presented at the annual 2010 to
2013 meetings of the Families of Spinal Muscular
Atrophy were searched by using the same key words,
and the Families of Spinal Muscular Atrophy Web site
(www.fsma.org) was carefully analyzed. Finally, a
selection of SMA clinical trials registered at clinical-
trials.gov has been included in the article.
RESULTS
Designing a Reasonable Clinical Trial for SMA:
Issues to Address
This section analyzes some of the issues and
concerns that need to be taken into account to design
a reasonable clinical trial for the treatment of SMA.
The increasing expansion of clinical trials planning
has raised several concerns, including the following:
are there enough patients identified to enroll easily?
What is the relative power of the study that can be
obtained? In addition, it is imperative to discern if
there are reliable, valid, and sensitive outcome meas-
ures available.12
Many difficulties exist in undertaking randomized,
double-blind, placebo-controlled studies of rare disor-
ders such as SMA. These difficulties include the process
of enrollment and stratification, even though interna-
tional registries for patients have increased the chances
of identifying and recruiting patients for clinical trials. In
terms of stratification, SMN2 copy number could be
used as a stratification criterion because the number of
copies of SMN2 correlates with motor function
achieved.13 Nevertheless, even in a relatively homo-
geneous group of SMA type I patients, 2 cohorts of
subjects can be identified: 1 characterized by an early
presentation (genetic diagnosis o6 months) and another
cohort with a relatively late onset of symptoms (genetic
diagnosis 46 months). Furthermore, another aspect that
makes the analysis of the data even more complicated is
that the intensity of care can profoundly affect
survival.14 Moreover, common standards of care, as
well as consistent assessment methods and outcome

Spinal Muscular Atrophy Drug Pipeline: Current

Basic
research FDA
seeds Preclinical: discovery Clinical deveopment
approval
ideas Identification Optimization Safety and manufacturing Phase I Phase II Phase III
IND
Trophos/olesoxime

ISIS - Biogen/ASO

Pfizer/quinazoline

NW/gene therapy

Genzyme, Sanofi/gene
therapy

PTC/Roche/small
molecule

Figure 1. List of the main clinical trials based on molecular strategies that have been conducted and are
ongoing, illustrated by their stage of development. IND ¼ Investigational New Drug; FDA ¼ US
Food and Drug Administration; Trophos ¼ Trophos SA; ASO ¼ antisense oligonucleotide; ISIS-
Biogen ¼ ISIS Pharmaceuticals/Biogen Idec; NW ¼ Nationwide Children’s Hospital; PTC/Roche ¼
Hoffmann-La Roche AG/PTC Therapeutics, Inc/SMA Foundation.

January 2014 129

 Clinical Therapeutics
measures to make data from different centers
comparable, are needed when designing a clinical trial.15
Drug selection process and study design are factors
that also need to be taken into consideration. Drug
selection based on preclinical data may not be a reliable
predictor of a therapeutic effect in patients. Thus, it is
important to define how preclinical models parallel the
disease in humans and which of the most predictive
outcomes are for human SMA. Another important point
to address is the correct evaluation of drug pharmaco-
kinetics in humans. For instance, it is fundamental to
demonstrate that the target compound induces SMN
protein expression in the required cell types in vivo in
humans, following what it does in preclinical models.
Furthermore, the majority of Phase I and II trials
have enrolled patients with SMA types II and III;
Phase III trial design is underdeveloped. Trial imple-
mentation has to contend with various challenges:
information on SMA populations is not complete,
natural history data are not always reliable, and the
differences in incidence and survival between coun-
tries are unknown.
Treatment in late stages of the disease could partly
explain past trial failures. Mouse studies found that
neonatal treatment is fundamental for efficacy, sug-
gesting that a limited therapeutic window exists, at
least in animal models.16 It has been suggested that the
first few months of life are the most critical time of
denervation in SMA types I and II, leading to the idea
that implementation of neonatal trials and the
development of strategies for identifying patients at
presymptomatic or early symptomatic stages would be
the best option for developing an efficient clinical trial
for SMA. Therefore, the timing of SMN rescue can be
different in patients with SMA type I compared with
SMA type II or III patients.
Another difficulty is the absence of validated
markers of disease progression. For SMA type I,
electrophysiologic measures such as motor unit num-
ber estimation (MUNE) and compound muscle action
potential (CMAP) may be better indicators of efficacy
than survival.17 Furthermore, biomarkers of treatment
effects are urgently needed. Currently, numerous
overlapping motor functional scales serve as clinical
end points. These should be tailored for presym-
ptomatic, early symptomatic, and chronic stages for
each SMA subtype.
Overall, the main recommendations for conducting
a reasonable clinical trial can be summarized as
130
follows: (1) testing of the preclinical effectiveness of
drugs in SMA animal models; (2) planning of the
feasibility of each phase of the trials; (3) harmonization
of the European regulatory processes for drug approval;
(4) targeting of the optimal therapeutic window for
SMA; and (5) analysis of biomarkers (measures of
upper limb function for nonambulant patients, muscle
biomarkers [MUNE/CMAP, muscle MRI, EIM] and the
SMA Foundation’s biomarker panel) (Table).
The balance of this review provides information on
the main clinical trials based on molecular strategies
that have been conducted and are ongoing (Figure 1).
Neuroprotection Strategy for SMA: Olesoxime
(TRO19622)
Olesoxime (TRO19622) is a small molecule with a
cholesterol-like structure that displays strong neuro-
protective properties.18 It has demonstrated ef-
fectiveness in keeping motor neurons alive in culture.
Preclinical in vitro studies have shown that the
compound promotes the function and survival of
neurons and other cell types under disease-relevant
stress conditions when interacting with the mitochon-
drial permeability transition pore (Figure 2A).
In October 2010, Trophos SA initiated a pivotal
efficacy and safety study of olesoxime, in  150 SMA
patients, in a program funded by the Association
Francaise contre les Myopathies in France. This
efficacy and safety study is a 24-month, Phase II,
multicenter, randomized, adaptive, double-blind,
placebo-controlled study in nonambulant patients
aged 3 to 25 years with SMA type II and type III.19
Recruitment of 165 patients with SMA was completed
in September 2011; no further patients can be
enrolled. Patients in the study are treated for 2
years, and results are expected before the end of
2013. Trophos has been granted orphan drug
designation for olesoxime for the treatment of SMA
by the US Food and Drug Administration (FDA). In
the European Union, olesoxime has been granted
Orphan Medicinal Product designation for SMA by
the European Commission.20 Randomization was 2:1
for olesoxime or matching placebo. The subjects
enrolled in this study received either a liquid
suspension formulation of olexosime (100 of 150
patients; 100 mg/mL at a dose of 10 mg/kg
administered orally once a day with food at dinner)
or a liquid suspension formulation of placebo (50 of
150) always administered once a day with food at
Volume 36 Number 1
January 2014

Table. Description of biomarkers, functional scales, and electromyography measures conventionally used in the spinal muscular atrophy (SMA)
field. SMA-MAP biomarkers: list of top 13 SMA motor function regressor markers, called SMA-MAP, in 2 SMA populations. Twenty-seven
analytes were selected for inclusion into a new biomarker panel, called SMA-MAP. The 13 analytes that regressed to motor outcomes of
SMA in both the BforSMA (Biomarker for SMA) study and PNCR NHS (Pediatric Neuromuscular Clinical Research Network History
Study) were included in the panel and are shown in the table. A description of the main functional scales and electromyography measures
conventionally used is provided.

Correlation to Modified
HFMS
Correlated
Outcome PNCR BforSMA
Variable Full Name Function Measures NHS P P

SMA-MAP biomarkers
COMP Cartilage oligomeric Cell proliferation, apoptosis, cell movement and Motor o0.001 o0.001
matrix protein attachment; binds strongly to calcium MUNE
Strength
AXL Receptor tyrosine Transduces signals from the extracellular matrix FVC o0.001 o0.001
kinase by binding growth factor GAS6 regulating: cell Motor
survival, cell proliferation, migration, and PQP
differentiation
CD93 Cluster of Cell to cell adhesion, clearance of apoptotic cells Motor o0.001 o0.001
differentiation 93 MUNE
PQC
Strength
PEPD Peptidase D Splits dipeptides with a prolyl or hydroxyprolyl CMAP o0.001 o0.001
residue in the C-terminal position; plays an Motor
important role in collagen metabolism MUNE
Strength
THBS4 Thrombospondin-4 Adhesive glycoprotein, mediates cell-to-cell and Motor o0.001 o0.001
cell-to-matrix interactions Mune
LUM Lumican Regulates: collagen fibril organization and CMAP o0.001 o0.001
circumferential growth, corneal transparency, Motor

C. Zanetta et al.
epithelial cell migration, and tissue repair MUNE
Strength
MB Myoglobin Myoglobin is the primary oxygen- FVC 0.001 o0.001
carrying pigment of muscles Motor
131

Strength
132

Clinical Therapeutics
Table (continued).

Correlation to Modified
HFMS
Correlated
Outcome PNCR BforSMA
Variable Full Name Function Measures NHS P P

DPP4 Dipeptidyl Antigenic enzyme expressed on the surface of Motor 0.001 o0.001
peptidase-4 most cell types associated with: immune MUNE
regulation, signal transduction, and apoptosis PQP
Strength
SPP1 Secreted Has a role in: biomineralization, bone Motor 0.002 o0.001
phosphoprotein 1, remodeling, immune response, chemotaxis,
also known as apoptosis, and cell activation
osteopontin
CHI3L1 Chitinase-3–like Catalyzes the hydrolysis of chitin; has a role in: Motor 0.01 o0.001
protein 1 Inflammation and tissue remodeling
CDH13 Cadherin 13, also Roles: mediation of intracellular signaling in Motor 0.039 0.001
known as H- vascular cells, regulation of cell growth, guiding
cadherin (heart) molecule in vascular and nervous system
APCS Amyloid P component Interacts with DNA and histones, scavenges Motor 0.041 o0.001
nuclear material released from damaged
circulating cells
LEP Leptin Inhibits appetite, acts as adiposity signal, Motor 0.058 o0.001
interacts with amylin, mediates the feeling of
satiety, promotes angiogenesis, promotes the
activity of lung surfactant, acts on bone
metabolism
Functional scales
— —
Volume 36 Number 1

MFM Motor function Quantitative scale that makes it possible to

measure measure the functional motor abilities of
patients affected by neuromuscular diseases;
whatever the diagnosis and the extent of motor
deficiencies, MFM allows us to: specify the

(continued)
January 2014

Table (continued).

Correlation to Modified
HFMS
Correlated
Outcome PNCR BforSMA
Variable Full Name Function Measures NHS P P

symptoms and the evolution of neuromuscular

diseases; objectivize the repercussion of the
therapeutic measures; direct the rehabilitation
and adaptation measures; facilitate
communication between the various persons in
charge of care; and select homogeneous
groups of patients in view of therapeutic trials
HFMS Hammersmith Functional motor scale that can be used to: — —
Functional Motor assess gross motor abilities of nonambulant
Scale children with SMA, monitor progression and
gross motor abilities over time
Electromyography
measures
CMAP Compound muscle The size of the evoked CMAP reflects the number — —
action potential of muscle fibers accessible from the stimulated
nerve and will be reduced by atrophy (from
whatever cause) by conduction block in the
peripheral nerve or at the neuromuscular
junction
MUNE Motor unit number They have been used to study the rate of motor — —
estimation unit loss in patients affected by neuromuscular
disorders

C. Zanetta et al.
FVC ¼ forced vital capacity; PQP ¼ PedQL quality of life, parent score; PQC ¼ PedQL quality of life, child score.
133
 Clinical Therapeutics

A B
α δ Generation of antisense
ADP + P oligonucleotides or
β α β α F morpholino (base pair
with an intronic splicing
ATP b Cytoplasm silencer), allowing
Y incorporation of exon 7 into
H SMN2-nRNA

Membrane
a
F
C
Injection into the
Environment cerebral ventricles
H H H and spinal cord of
an SMA mouse

6 7 8
Exon 7 inclusion

Olesoxime Increased:
• SMN protein levels
• Size and strength of muscle fibers
• Number of spinal cord motor neurons
• Median survival

C
SMN2 DNA

Dcp1
Dcp2

SMN2
Pre-mRNA
No drug With quinazolines Quinazolines
Introns

SMN2
6 7 8 6 7 8
mRNA

Exons
6 8 6 7 8

Figure 2. Mechanisms of action of main molecular therapeutic strategies that are currently tested in clinical
trials for spinal muscular atrophy (SMA). (A) Olesoxime targets proteins of the outer mitochondrial
membrane and prevents permeability transition pore opening mediated by, among other things,
oxidative stress. (B) Antisense oligonucleotides (ASOs), with different chemical structures including
morpholino chemistry, designed to target and block intronic splicing silencer number one (ISSN-1)
are able to promote exon 7 inclusion in the majority of survival motor neuron gene (SMN2) mRNA
transcripts and therefore lead to the production of full-length and functional SMN protein. (C)
Quinazolines are a family of compounds that inhibits RNA decapping enzymes (DcpS; Dcp 1 and 2
are represented in the figure) involved in RNA turnover. This inhibition can consequently increase
full-length SMN2 transcript and SMN protein.

dinner. The primary end point of the study is the
change from baseline in the Motor Function Measure
(MFM) functional scale. Secondary end points include
the Hammersmith Functional Motor Scale (HFMS)
and electromyography (CMAP and MUNE) as well as
measures of safety, tolerance, and quality of life. The
study is being conducted at 22 centers in 7 European
countries. Inclusion criteria include weakness and
hypotonia consistent with a clinical diagnosis of
SMA type II or III and laboratory documentation of
homozygous absence of SMN1 exon 7 and/or deletion
and mutation on other alleles. To be included, subjects
had to have an MFM relative score (percentage of the
maximum sum of both dimensions) Z15% (D1 þ D2
score) and HFMS score at baseline Z3. The study
enrolled nonambulant patients, defined as patients
with an HFMS score r38, aged 3 to 26 years at
time of enrollment. The patients’ age at onset of

134 Volume 36 Number 1


symptoms had to be r3 years. Additional inclusion
criteria were normal laboratory test results and the
ability to take the study treatment. Exclusion criteria
were significant comorbidities and administration of
other medications intended for SMA treatment. The
ongoing study period was divided into 3 stages: stage
1, three-month safety assessment to confirm the
adequate dose after 1 month of treatment (an inde-
pendent data monitoring committee [DMC] has the
goal of assessing the safety of olesoxime every 3
months); stage 2, efficacy/futility analyses at 1 year
(a first-interim efficacy analysis was planned; after
that, all patients will be treated for 1 year [52 weeks]
to assess the need to continue the study to reach the
planned objectives); and stage 3, efficacy and safety
analysis at 2 years.
Trophos announced the completion of the interim
analysis of the pivotal efficacy study of olesoxime. The
independent DMC has reviewed the treatment effects
at 1 year, taking into account the primary outcome
measures of efficacy and changes in motor function
(MFM scale), along with the latest safety report
including ECG tracings, periodic laboratory findings,
hemostatic parameters, and serious adverse event
listings for all participants. Based on the trial-
stopping criteria as defined in the protocol, as well
as the absence of safety concerns related to olesoxime
treatment, the DMC recommendation is to continue
the study as planned. Following the recommendations
of the DMC, the study will continue until all partic-
ipants are treated for 2 years, with finalization of the
last patient scheduled for September 2013. Top-line
results are expected by the end of 2013.20,21
Oligonucleotides Therapy for SMA: ISIS-SMNRx
SMA is caused by the loss of the SMN1 gene,
leading to reduced SMN protein levels. The human
genome harbors an additional SMN2 gene (or genes)
that produces low levels of full-length SMN but
cannot adequately compensate for the loss of SMN1
due to an aberrant splicing. The majority of SMN2
gene transcript lacks exon 7 and the resultant SMNΔ7
mRNA is translated into an unstable but partially
functional protein. Therapeutic strategies with the
goal of promoting exon 7 retention, thus increasing
full-length SMN2 transcript, are promising treatments
for patients with SMA. Different splice-silencing se-
quences in SMN2 have been identified as potential
targets for splice modifications directed by antisense
January 2014
C. Zanetta et al.
oligonucleotides. A particularly strong splice silencer
is found in SMN2 intron 7 downstream of exon 7.
Antisense oligonucleotides designed against this motif
promote SMN2 exon 7 retention in the mature SMN2
transcripts, increasing SMN protein expression in
SMA cells (Figure 2B).22
Isis Pharmaceuticals is developing a specific oligo-
nucleotide called ISIS-SMNRx designed to act against
this strong splicing silencer to promote the inclusion of
the missing exon (exon 7); the goal is to create an
mRNA that codes for the production of the full-length
functional SMN protein for proper motor neuron
function. The FDA granted orphan drug status and
fast track designation to ISIS-SMNRx for the treatment
of patients with SMA. Isis is currently in collaboration
with Biogen Idec to develop and potentially commerci-
alize this compound to treat all types of SMA. Under
the terms of the January 2012 agreement, Isis is
responsible for global development and Biogen Idec
has the option to license the compound until com-
pletion of the first successful Phase II/III study. Several
nonprofit organizations (including Families of Spinal
Muscular Atrophy) also support this study. ISIS-
SMNRx is a uniformly 2′-O-methoxyethyl modified
antisense drug that corrects the splicing disorder in
SMN2, resulting in the production of fully functional
SMN protein in model systems. In mild and severe
SMA mouse models, it is able to provide both a
phenotypic and a pathologic benefit when delivered
centrally23,24 and has a long half-life in central
nervous system tissue (46 months in animal models).
The results in preclinical studies were very promising,
with rescue of the SMA phenotype with early systemic
treatment.25
Isis has completed a Phase I, open-label, safety,
tolerability, and dose range–finding study with the
purpose of testing the safety, tolerability, and phar-
macokinetics of a single dose of ISIS-SMNRx admin-
istered into the cerebrospinal fluid (CSF) as a single
injection in patients with SMA.26 This study was
started in November 2011 and was completed in
January 2013. A total of 28 subjects were enrolled
in this trial, and all of them completed the study; no
healthy volunteers were accepted. The inclusion criteria
of this study were: a documented SMN1 homozygous
gene deletion and clinical signs attributable to SMA, age
2 to 14 years at time of screening, the ability to complete
all study procedures, and the demonstration that
parents/patients have adequate supportive psychosocial
135
 Clinical Therapeutics
circumstances. Moreover, the estimated life expectancy
had to be 42 years from the time of screening. The
major exclusion criteria were: respiratory insufficiency
defined by the need for invasive or noninvasive
ventilation during a 24-hour period, presence of a gastric
feeding tube, previous scoliosis surgery or scoliosis
surgery planned during the duration of the study that
would interfere with the lumbar puncture injection
procedure, conditions that interfere with intrathecal
administration of the drugs, severe comorbidities, or
taking medications aimed at treating SMA. Four dose
levels have been sequentially evaluated. Each dose level
was studied in a cohort of 6 or 10 patients; all patients
received active drug. Primary end points of the study
were safety/tolerability and CSF and plasma drug level
pharmacokinetics. Safety and tolerability assessments
were obtained through analysis of adverse events,
neurologic examinations, CSF laboratory tests, vital
signs, clinical laboratory tests, physical examinations
and weight reports, ECGs, and the analysis of concom-
itant medication use. Pharmacokinetic measures used
were plasma levels of the drug (over 24 hours’ postdos-
ing and at 7 days’ postdose) and CSF levels of drug (at 7
days’ postdose).
Exploratory end points of this study of ISIS-SMNRx
were HFMS and neuromuscular electrophysiology
CMAP/MUNE.26 Safety and tolerability results can
be summarized as follows: (1) ISIS-SMNRx was well
tolerated, with no significant safety finding when
given as a single dose up to 9 mg; (2) the injection
procedure was well tolerated and was shown to be
feasible; and (3) data suggest that the tolerability of
the injection procedure is improved with the use of a
smaller needle (ie, 24 or 25 gauge). In terms of drug
concentrations, CSF and plasma drug levels were
measurable, and they were dose dependent and only
moderately variable. Through the use of HFMS, it
was evaluated that at day 85, there was a mean
change in motor function from baseline of 3.1 points
(P ¼ 0.02) and a percentage of change of 17.6%.
Moreover, 6 of 10 subjects had a change from baseline
major of 4 points from their basal level score at the
HFMS (3 of 6 of these subjects were aged 45 years).
CMAP and multipoint incremental MUNE were per-
formed in the highest-dose group at baseline and
at day 85, with the primary purpose of determine the
feasibility of using this method in patients with SMA.
These examinations were performed on the right
ulnar nerve that innervates the abductor digiti minimi.
136
Electrophysiology measurements demonstrated stable
CMAP, with a potential increase in MUNE. The
conclusions and implications of this Phase I study are
that ISIS-SMNRx was well tolerated at all dose levels,
and no safety or tolerability concerns were identified.
The injection procedure used was shown to be feasible
in children with SMA. CSF and plasma drug concen-
trations observed were dose dependent and consistent
with preclinical data, supporting infrequent administra-
tion (ie, every 6–9 months). An improvement in HFMS
scores was observed in the group injected with the
highest dose. Electrophysiology measurements in the
highest-dose cohort at 3 months demonstrated an
increase in MUNE with stable CMAP.
In September 2013, the preliminary results of the
open-label Phase I study of ISIS-SMNRx in children
with SMA (24 subjects) were reported; they high-
lighted that the majority of children with SMA
receiving the 2 highest doses of the drug (6 and 9
mg) continued to show improvements in muscle
function tests (mean HFMS improvement with a dose
of 9 mg of ISIS-SMNRx : 5.75) up to 14 months after a
single injection of the drug.21,27 The amelioration was
dose dependent, and no decline was observed.
Although the data are encouraging, it must be empha-
sized that no placebo was included in the study. Based
on this evidence, the investigators have amended the
infant study to increase the dose from 9 to 12 mg.28 In
addition to the infant pilot study, Isis is also
completing a multiple-dose, dose-escalating Phase Ib/
IIa study of ISIS-SMNRx in children with SMA type II
and III. Isis has completed dosing in all 3 dose cohorts
(3, 6, and 9 mg) and is now considering adding a 12-
mg dose cohort to this study.29
ISIS-SMNRx is now being studied in a multiple-
dose Phase II study27,30 that is designed with the goal
of testing the safety, tolerability, and pharmacoki-
netics of multiple doses of the drug administered into
the spinal fluid 3 times over the duration of the trial in
patients with infantile-onset SMA. Two dose levels
will be evaluated sequentially. Each dose level will be
studied in a cohort of 4 patients; all patients will
receive active drug.30 The estimated number of
subjects enrolled in the study is 8, and the eligible
age is up to 210 days. No healthy volunteers have
been accepted. The inclusion criteria are: genetic
documentation of 5q SMA (homozygous gene dele-
tion or mutation), onset of clinical signs and symptoms
consistent with SMA at Z21 days and o6 months
Volume 36 Number 1

(180 days) of age at study entry, receiving adequate
nutrition and hydration, body weight 45th percentile
for age according to Centers for Disease Control and
Prevention guidelines, medical care that meets and is
expected to continue meeting guidelines set out in the
Consensus Statement for Standard of Care in Spinal
Muscular Atrophy6 in the opinion of the site investi-
gator, gestational age of 35 to 42 weeks, and gesta-
tional body weight Z2 kg. Moreover, patients need to
reside within  9 hours’ ground-travel distance from a
participating study center for the duration of the study
(residence 42 hours’ ground-travel distance from a
study center can require special clearance).30
Major exclusion criteria are: hypoxemia (oxygen
saturation awake o96% or oxygen saturation asleep
o96%, without ventilation support) and significant
comorbidities, in particular those interfering with CSF
administration, or treatment with another investiga-
tional drug. The main primary outcome measure of
the study is the number of participants with adverse
events (participants will be followed up for the
duration of the study). Secondary outcome measures
are: (1) plasma pharmacokinetics (assessments at 1, 2,
4, and 24 hours after dosing); (2) Cmax; (3) Tmax; and
(4) the AUC from the time of the intrathecal dose to
the last collected sample (20 hours after dosing).30
The infantile-onset study might provide important
information related to the therapeutic windows of
SMN rescue.
Small Molecules for SMA: Quinazolines
Quinazolines are a family of compounds that
inhibits RNA decapping enzyme DcpS (involved in
RNA turnover). This inhibition can consequently
increase SMN2 expression (Figure 2C).31 A high-
throughput screening identified a few quinazolines as
lead compounds that increase full-length SMN2 tran-
script and SMN protein.32 These compounds had
poor blood–brain barrier (BBB) penetration and
required high doses to achieve a sufficient upregula-
tion of SMN. Based on a lead quinazoline compound,
derivatives were developed to overcome the obstacles
associated with the low BBB penetration. Further
optimization of this compound led to the identifi-
cation of D157495, also called RG3039, which
ameliorates the phenotype of SMA mice.16,33
Currently, RG3039 is undergoing a Phase I clinical
trial in which the safety and efficacy of various doses
will be evaluated.21 This compound is included in
January 2014
C. Zanetta et al.
the Pfizer SMA Drug Development Programs as
PF-06687859. A Phase I, first-in-man, double-blind,
placebo-controlled, ascending single-dose, safety and
pharmacokinetics study in healthy volunteers has been
conducted. Two of 4 cohorts of a multidose Phase I
study in 16 healthy volunteers have been completed,
with no further patient enrollment. A biomarker and
optimal dosing plan is being generated, as well as
backup compounds, and enabling technologies have
been developed.
Planned Clinical Trials
Gene Therapy
Gene therapy promises a permanent solution for
SMA through viral delivery and insertion of the entire
SMN1 gene or cDNA sequence into the genome of
patients with SMA.31 This process is generally
irreversible, and there are apparent risks if the
exogenously delivered gene is inserted at a wrong
location and/or overexpressed. Overall, the results of
gene therapy aiming at SMN1 delivery by scAAV9 in
SMA mice and large animals seem promising and may
offer one of the best therapeutic alternatives to a
specific group of SMA patients who are too weak to
receive frequent invasive treatments.
After submission of an Investigational New Drug
application, the FDA has given its approval to
physician/scientists at Nationwide Children’s Hospital
in Columbus, Ohio, to begin a Phase I clinical trial of
a systemic AAV9-delivered human SMN gene.21 The
clinical trial is expected to begin in early 2014 and will
be limited to patients with type I SMA, with an age
range of 0 to 9 months. Previous research from
Nationwide Children’s Hospital Principal Investiga-
tor Brian Kaspar, PhD, demonstrated that the AAV9
viral vector is able to cross the BBB. Based on these
findings and additional preclinical studies, the SMN
gene will be delivered by injection into the blood-
stream as part of this Phase I trial. Neurologist Jerry
Mendell, MD, director of the Center for Gene
Therapy at Nationwide Children’s Hospital, will
lead the study. They plan to recruit 9 subjects, with
an age of r6 months with proven SMN mutation and
retaining 2 copies of SMN2. Exclusion criteria are the
use of ventilatory support or pulse oximetry satura-
tion o95% and high levels of antibody directed
against AAV9.
Physician/scientists are also planning a dose esca-
lation study (low and high dose). The improvement
137
 Clinical Therapeutics
function will be determined by SAFETY/CHOP-IN-
TEND (Children's Hospital of Philadelphia Infant
Test of Neuromuscular Disorders). The planned
follow-up is at 1, 2, 4, 8, and 12 weeks and then
every 3 months until 2 years of age. An additional
study is planned using the AAV9-delivered SMN gene,
with the aim of examining a different route of
administration (injecting the virus into the CSF). The
safety study has been completed in large mammals,
and the study is proceeding toward the pre–Investiga-
tional New Drug stage at the FDA.
Moreover, Genzyme/Sanofi are planning a SMN1
gene replacement therapy program. They are prepar-
ing to nominate a vector candidate for preclinical
toxicology studies and for studies in nonhuman
primates to optimize delivery protocol and dose.
Overall, gene therapy is one of the most promising
new approaches, given its potential effectiveness in
resolving the SMA molecular defect.21
Small Molecules
Small molecule compounds aimed at increasing
SMN levels offer several advantages, including an
easy transport across biological barriers. Considering
that SMA is a neurodegenerative disease, compounds
that are transported across the BBB would be best
suited for an effective therapy.
Small molecule SMN2 splicing modifiers, which
increase full-length SMN mRNA and protein in cells
isolated from SMA patients, are being developed by
Hoffmann-La Roche AG/PTC Therapeutics, Inc/SMA
Foundation.21 In August 2013, PTC Therapeutics
announced the selection of a development candidate.
This approach could be advantageous if an easy
administration route (eg, oral administration) is
possible. However, this strategy may be limited by
the possibility of off-target effects.
CONCLUSIONS
Currently, no effective treatment is available for SMA
and other connected motor neuron disorders. The
main therapeutic strategies that are now in use are
based on symptomatic treatment and supportive care.
At a preclinical level, many therapeutic strategies have
been investigated, and some of them have been tested
and continue to be tested in clinical trials both in
Europe and in the United States. This review outlined
the principal aspects that should be taken into account
when designing a reasonable clinical trial. Moreover,
138
we also provided a description of the most promising
clinical trials that are currently ongoing and/or are
planned for the near future.
The overall evaluation is that an increasing num-
ber of clinical studies over the next few years are
expected for SMA. One issue that is noteworthy and
unique for SMA is that the phenotype of patients
varies significantly among different SMA types (I, II,
and III). Moreover, even in a relatively homogenous
group of SMA type I patients, 2 cohorts of sub-
jects can be identified: 1 characterized by an early
presentation (genetic diagnosis o6 months) and
another 1 with a relatively late onset of symptoms
(genetic diagnosis 46 months). This aspect cannot
be ignored when planning a reasonable clinical trial
for SMA. SMN2 copy number directly correlates
with both clinical phenotype and highest motor
function achieved by patients and could therefore be
used as a stratification criterion. Therefore, a power-
ful strategy could be to design different clinical trials
according to each SMA subtype. Although many
therapeutic strategies for SMA (eg, stem cell therapy)
are being developed, what now seems certain is that
more preclinical studies are needed before those
strategies could be investigated in clinical trials and
therefore used for the treatment of SMA in human
patients.
Overall, the results of both preclinical and early
clinical trials involving novel molecular therapies
suggest that the clinical care paradigm in SMA will
soon change.
ACKNOWLEDGEMENTS
This work was supported by the Ministry of Health
(GR-2009-1483560) and MIUR (FIRB RBFR08RV86)
to Dr. Corti. The authors also thank the “Associazione
Amici del Centro Dino Ferrari” for their support. Dr.
Zanetta was responsible for the study design, literature
search, figure creation, writing, main revision. Dr.
Monguzzi was responsible for the literature search
and revision. Dr. Corti was responsible for the study
design, literature search and writing. All remaining
authors contributed equally in the literature search
and revision processes.
CONFLICTS OF INTEREST
The authors have indicated that they have no conflicts
of interest regarding the content of this article.
Volume 36 Number 1

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Address correspondence to: Stefania Corti, MD, PhD, IRCCS Foundation

Ca’ Granda Ospedale Maggiore Policlinico, via Francesco Sforza 35,
20122 Milan, Italy. E-mail: stefania.corti@unimi.it

140 Volume 36 Number 1

©2014 Elsevier