J Biosci (2024)49:36 Ó Indian Academy of Sciences

DOI: 10.1007/s12038-023-00412-9 (0123456789().,-volV)

(0123456789().,-volV)

Review
Spinal muscular atrophy: Molecular mechanism of pathogenesis,
diagnosis, therapeutics, and clinical trials in the Indian
context

ASHUTOSH AASDEV1,2, SREELEKSHMI R S 1, V RAJESH IYER1 and

SHIVRANJANI C MOHARIR1*
1
Tata Institute for Genetics and Society, Bengaluru, India
2
WNS Triange, WNS Global, Bengaluru, India

*Corresponding author (Email, shivranjani.c@tigs.res.in)

MS received 6 September 2023; accepted 5 December 2023

Spinal muscular atrophy (SMA) is a neuromuscular, rare genetic disorder caused due to loss-of-function
mutations in the survival motor neuron-1 (SMN1) gene, leading to deficiency of the SMN protein. The severity
of the disease phenotype is inversely proportional to the copy number of another gene, SMN2, that differs from
SMN1 by a few nucleotides. The current diagnostic methods for SMA include symptom-based diagnosis,
biochemical methods like detection of serum creatine kinase, and molecular detection of disease-causing
mutations using polymerase chain reaction (PCR), multiplex ligation-dependent probe amplification (MLPA),
and exome or next-generation sequencing (NGS). Along with detection of the disease-causing mutation in the
SMN1 gene, it is crucial to identify the copy number of the SMN2 gene, which is a disease modifier.
Therapeutic options like gene therapy, antisense therapy, and small molecules are available for SMA, but, the
costs are prohibitively high. This review discusses the prevalence, diagnosis, available therapeutic options for
SMA, and their clinical trials in the Indian context, and highlights the need for measures to make indigenous
diagnostic and therapeutic interventions.

Keywords. Gene therapy; neuromuscular; rare genetic disorder; spinal muscular atrophy; survival motor
neuron

1. Introduction 2009; Sugarman et al. 2012). The incidence of 1 in

6,000 to 1 in 10,000 live births and carrier frequency
Spinal muscular atrophy (SMA) is an autosomal ranging from 1 in 25 to 1 in 209 individuals have been
recessive, neurodegenerative disease and the second reported for SMA in populations of different regions
leading genetic cause of deaths in infants throughout (Ben-Shachar et al. 2011; Sangaré et al. 2014; Verhaart
the world (Lunn and Wang 2008). SMA is character- et al. 2017; Chen et al. 2020; Verma et al. 2020; Nilay
ized by progressive degeneration of motor neurons in et al. 2021).
the anterior horn of the spinal cord, leading to skeletal SMA is a debilitating disease, not only for the
muscle weakness and atrophy (Burr and Reddivari individual but for the family as well. Patients with type
2022). The incidence and carrier frequency of SMA II or III SMA need supportive care for a long time.
varies in different ethnic groups (Hendrickson et al. Families may not be always able to afford them or
access to such care may be limited. Physiotherapy is
recommended for many patients but economic prob-
This article is part of the Topical Collection: The Rare lems, lack of family support, and travelling difficulties
Genetic Disease Research Landscape in India. are common obstacles for patients (Bose et al. 2019).

http://www.ias.ac.in/jbiosci
36 Page 2 of 17 A Aasdev et al.
Even with treatment options being available, patients
cannot rest easy, as the costs for these are colossal,
amounting to tens of crores in Indian Rupees (INR),
which is not affordable to most patients. For example,
an injection of nusinersen, an antisense oligo therapy
for SMA, costs around 125,000 USD or around 1 crore
INR. The total cost of the injection in the first year is
round 6.2 crores INR, and 3.1 crores INR for subse-
quent years (Suthar and Patil 2021). The cost of the
SMA gene therapy, Zolgensma, marketed by Novartis,
is around 14 crores INR. The third drug, risdiplam, a
small-molecule drug, is approved by the Drug Con-
troller General of India (DCGI) and one vial costs
around 6 lakhs INR. Nusinersen is imported in India
for only a few patients through the Individual Patient
Humanitarian Access Program. Zolgensma or AVXS-
101 is also accessed through humanitarian programs.
Similarly, risdiplam is made available in India by
Roche through a compassionate program. These drugs
are, however, available only to a very few selected
patients. Another problem associated with the pro-
curement of these drugs through humanitarian pro-
grams is the associated paperwork. In many instances,
by the time the paperwork is done, the patient either
crosses the therapeutic window or succumbs, especially
in case of SMA type I. Government interventions to
help spread the awareness and reach of these treatments
and generic manufacturing through license agreements
are required to help patients (Suthar and Patil 2021).
2. Incidence of SMA in India
India has a population size of more than 1.4 billion,
and a diverse group of communities. Endogamy,
cultural boundaries, and founder effects over a long
period of time have resulted in the accumulation of
many genetic variations and diseases (Bajaj et al.
2019). The exact incidence of SMA in India is not
known. However, there have been a few cohort
studies in which the incidence of SMA in different
regions in India is estimated. Verma et al. in 2020
analysed 200 couples or 400 non-consanguineous
subjects from a North Indian population, and found
that 9 of them had a deletion of one SMN1 gene
(Verma et al. 2020). They reported the carrier fre-
quency to be 1 in 44 in their cohort. They also
indicated that SMA is common in India and that it is
the top third genetic disorder, after b-thalassemia and
Duchenne muscular dystrophy. Between the years
1997–2020, they ran 2256 diagnostic tests and 1052
prenatal diagnoses for SMA. They also suggested that
assuming Hardy–Weinberg equilibrium, the incidence
of SMA can be around one in 7744* births. In the
same year, in 2020, another group published the
carrier frequency of SMA to be 1 in 38 in the state of
Uttar Pradesh in India, that houses around 16.5% of
the Indian population according to the 2011 census
data (Nilay et al. 2021). They had analysed 606
individuals between the year 2016–2019 and found 16
of them to be carriers for SMA. After applying the
Hardy–Weinberg principle, they estimated the inci-
dence of SMA to be one in 5620* live births. A more
recent study published in 2022 found the carrier fre-
quency of the heterozygous deletion of the SMN1
gene to be 1 in 30 among individuals in the repro-
ductive age in their cohort of 198 subjects from the
North Indian states of Punjab, Jammu & Kashmir, and
Chandigarh (Kaur et al. 2022). They estimated the
incidence of SMA to be 1 in 3333* live births, by
applying the Hardy–Weinberg principle. Based on
these studies and the current birth rate (79,726 live
births average per day; https://countrymeters.info/en/
India) in India, it can be deduced that the incidence of
SMA in India is approximately 10–24* births every
day! Annually, 3600 to 8640 newly born infants can
plausibly be affected by SMA.
3. Classification of phenotypes of SMA
SMA pathology involves the key feature of progres-
sive loss of motor neurons in the spinal cord and
brainstem. SMA has been divided into five clinical
types based on the age of onset and motor functions
affected (figure 1) (Prior et al. 1993). Type 0 SMA,
the most severe form, is presented prenatally in the
foetus. After birth, type 0 infants require respiratory
aid and have severe muscle weakness and hypotonia.
Even with supportive care, these infants do not sur-
vive past 6 months from birth. Type 1 SMA, also
known as Werdnig–Hoffmann disease is acute, severe,
and the most common form of the disease, accounting
for about 80% of cases (Emmady and Bodle 2022).
The onset of symptoms is before 6 months of age, the
mean age being 2.5 months, and death occurs within
the first 2 years. The diaphragm is not affected, but
the intercostal muscles are weak, leading to a bell-
shaped upper body and paradoxical breathing char-
acteristic of infantile SMA. They have a weak muscle
*
Without considering the effect of SMN2 on the phenotype and
the 2/0 genotypes, explained in the following sections.
 Indian clinical and research scenarios for SMA
Figure 1. Different types of SMA, age of onset, symptoms, SMN2 copy number, and life expectancy.
tone and lack motor development. Type II SMA, also
known as Dubowitz disease, manifests between 7 and
18 months of age, with a mean age of 8.3 months,
and the survival age of the patients, with protective
care, is until early adulthood (Cancès et al. 2020).
The disease is of intermediate severity with the
affected individuals having some motor control, which
is lost as the disease progresses. Respiratory insuffi-
ciency is the major cause of morbidity due to pro-
gressive respiratory muscle weakness. Type III SMA,
also known as Kugelberg–Welander disease, has age
of onset after 18 months. These individuals usually
live a normal lifespan with supportive care. Their legs
are often weaker than their arms. Some can sit, stand,
and even walk with no aid, while others require aid to
walk properly. Motor function slowly declines with
early adulthood and ambulation may be lost in
adulthood, depending on the onset of symptoms.
Patients affected earlier succumb in the teens, while
those with late onset lose their ambulatory ability in
their thirties (Salort-Campana and Quijano-Roy 2020).
Type IV SMA or adult-onset SMA affects less than
5% of SMA patients. The age of disease onset is the
second decade or later, with individuals living a
normal lifespan. Respiratory function is normal.
Individuals have motor impairment only after their
third decade. Complications often seen with SMA are
restrictive lung disease, scoliosis, and nutritional
impairment, for which they require supportive care
(Prior et al. 1993; Souza et al. 2021).
Page 3 of 17 36
4. Genetic basis of SMA
Spinal muscular atrophy is a genetic disorder inherited
in an autosomal recessive manner. The diverse clinical
manifestations between SMA types I, II, and III ini-
tially led to questioning the possibility of genetic
heterogeneity between the different types. Break-
through studies in 1990 identified the genetic loci
responsible for chronic SMA (types II and III) to be
located on the long arm of chromosome 5, between
5q11.2 and q13.3 (Brzustowicz et al. 1990). This was
also confirmed by another group in the same year
(Melki et al. 1990). Later on, the locus for acute SMA
(type I) was also mapped to the same genomic region
of 5q11.2–q13.3 (Gilliam et al. 1990). This ruled out
the possibility of genetic heterogeneity as the reason
for the phenotypic of SMA diversity and proved that
SMA is indeed caused due to mutations at the same
locus. The region responsible for SMA was fine-map-
ped over the years, with the complexity in the region,
such as polymorphisms, duplications, and pseudoge-
nes, becoming apparent (Soares et al. 1993; Thompson
et al. 1993; Morrison 1996). The gene responsible for
SMA was identified in 1995 by Lefebvre et al. (1995)
and was called the survival motor neuron (SMN) gene,
after screening the human foetal cDNA library with
candidate region probes. As the chromosomal region is
an inverted duplication, two copies of SMN were
identified on chromosome 5. The one towards the
telomeric region was called SMN, later changed to
36 Page 4 of 17 A Aasdev et al.
SMN1. The other centromeric proximal copy was
called cBCD541, which was later changed to SMN2.
Both copies of the gene differ only by a few nucleotide
bases. Another gene, neuronal apoptotic inhibitory
protein (NAIP), which is mapped to the SMA locus, is
also found to be disrupted in many SMA individuals
and is believed to be a disease modifier that contributes
to the SMA phenotype (Roy et al. 1995; Zhang et al.
2020). Further studies indicated that point mutations in
SMN1, deletions in any of the exons or large deletions
encompassing SMN1 are observed in patients with
SMA (Lefebvre et al. 1995; Wirth et al. 1995; Rodri-
gues et al. 1996; Butchbach 2021). The involvement of
SMN2 in the SMA phenotype was hypothesized by
Lefebvre et al. (1995). A total of 98.6% of SMA
individuals in their study cohort of size n = 229 had
disruptions in the SMN1 copy, while SMN2 was intact.
They also observed gene conversion of SMN1 to
SMN2. This observation was further confirmed in other
studies highlighting higher prevalence of SMN1 dele-
tions in acute SMN1 with gene conversion to SMN2 in
chronic or mild SMN2 cases (Burghes 1997; DiDonato
et al. 1997b; McAndrew et al. 1997; Parsons et al.
1998). Another important finding came in the form of
de novo or sporadic cases of SMA in children whose
parents were not affected or were carriers. Such cases
account for about 2% of SMA cases (Wirth et al.
1997).
It is now known that the SMA critical region on
chromosome locus 5q is a 500 kb inverted duplicated
region unique to humans (Rochette et al. 2001). The
region has four genes SERF1A, SMN1, NAIP, and
GTF2H2 and their duplicated copies SERF1B, SMN2,
WNAIP D5 (pseudogene NAIP with exon 5 deletion),
and WGTF2H2B (GTF2H2 pseudogene) (figure 2).
Nearly 95% of SMA cases are caused due to
homozygous deletion of SMN1 or conversion of the
SMN1 gene to SMN2, while SMN2 is not affected.
SMN1 and SMN2 both contain nine exons (1, 2a, 2b,
3–3) and span a *30 kb region. In general, both the
genes differ by only one nucleotide, SMN2 c.850C[T
substitution, a silent mutation in the coding region
(Monani et al. 1999). The base ‘C’ at the sixth position
in exon 7, in SMN1, has been determined as an exon
splicing enhancer (ESE) sequence which facilitates the
inclusion of exon 7 in full-length SMN1 transcripts,
leading to the formation of the functional SMN protein
(Lorson et al. 1999; Monani et al. 1999; Lorson and
Androphy 2000). However, the transition from C to T
at this sixth position in exon 7 in SMN2 disrupts the
ESE, leading to a majority of alternative spliced SMN2
transcript isoforms that exclude exon 7 and form a
mutant SMN protein (SMND7) (Cartegni and Krainer
2002). The base ‘T’ at the sixth position in exon 7 in
SMN2 creates an exon splicing suppressor (ESS)
instead of an ESE, which promotes exclusion of exon 7
and expression of the mutant SMN protein (Kashima
and Manley 2003; Kashima et al. 2007). It has been
proposed that the effect mediated by ESS antagonises
the effect of ESE, leading to exclusion of exon 7
(Cartegni et al. 2006). A detailed overview of the
splicing mechanisms has been extensively discussed by
Singh and Singh (2018).
5. Phenotypic manifestations caused due to genetic
variability of SMN locus
SMA disease is caused due to a lack of SMN protein
which leads to degeneration of motor neurons in the
spinal cord. Disease severity is inversely correlated
with SMN protein levels (Lefebvre et al. 1997;
Crawford et al. 2012). The SMN protein is expressed
throughout the somatic tissues and is localized in
nuclear structures called gems. SMN is necessary for
the assembly and function of small nuclear ribonucle-
oprotein (snRNP) complexes and messenger ribonu-
cleoprotein (mRNP) complexes which are required for
the splicing of pre-mRNA and mRNA transport along
axons (Liu et al. 1997; Pellizzoni 2007; Burghes and
Beattie 2009). Thus, reduced amount of SMN protein
hampers snRNP assembly and in turn changes the
amounts of mRNA, causing altered levels of proteins
required for normal cell growth. Normal SMN is an
oligomeric protein. It has been suggested that in the
SMND7 protein, self-association is defunct, thus
inhibiting SMN function (Lorson et al. 1998). The
reason why motor neurons are affected more than other
cell types, even though the protein is ubiquitously
expressed, is still unclear.
Functional SMN protein is produced by the SMN1
gene. Homozygous disruptions in SMN1 are responsi-
ble for the clinical symptoms seen in SMA patients.
SMN2 is intact in SMA individuals and predominantly
produces the mutant SMND7 protein, which is unsta-
ble and is degraded in cells through the ubiquitin
proteasomal system (Burnett et al. 2009). Almost 20%
of SMN2 still produces complete transcripts and the
functional SMN protein (Cho and Dreyfuss 2010). It is
also known that SMN2 is unique to Homo sapiens and
varies between 0 and 8 copies in the human genome
(Rochette et al. 2001). Since, SMN2 produces some
 Indian clinical and research scenarios for SMA Page 5 of 17 36

Figure 2. Image showing the SMA critical region on chromosome 5: The SMA critical region on chromosome 5q13
consists of four genes, SERF1A, SMN1, NAIP, and GTF2H2, and their duplicated copies, SERF1B, SMN2, WNAIP D5
(pseudogene NAIP with exon 5 deletion), and WGTF2H2B (GTF2H2 pseudogene). Almost 95% of SMA patients have
deletion of exon 7 of the SMN1 gene or conversion of SMN1 to SMN2. SMN1 and SMN2 differ by a single nucleotide in the
coding in region – the ‘C’ in exon 7 in SMN1 is replaced by ‘T’ in SMN2, leading to skipping of exon 7 in most of SMN2
transcripts. Very few complete transcripts, without the skipping of exon 7, are also formed from SMN2, which results in
production of minute amounts of the functional SMN protein from SMN2.

functional SMN protein, higher SMN2 copy number is
associated with a milder disease phenotype (Elsheikh
et al. 2009; Calucho et al. 2018; Sun et al. 2020).
Mouse and zebrafish have only one SMN gene which is
an ortholog of human SMN1 (DiDonato et al. 1997a).
Homozygous SMN deletion in mice is embryonically
lethal. This loss of SMN can be partially rescued by
complementation with two copies of human transgenic
SMN2. However, the mice develop a severe neurode-
generation phenotype resembling type I SMA and die
within a few days of birth (Monani et al. 2000). Copy
number of 3–4 of human transgene SMN2 in SMN-/-
mice, leads to development of a milder SMA pheno-
type, while transgenic mice with 8 copies of human
SMN2 are phenotypically normal (Monani et al. 2000;
Michaud et al. 2010). Similarly, SMN-null zebrafish
can also be rescued by complementation with human
SMN2 (Hao et al. 2011). Bulk SMND7 along with the
functional SMA protein in minor amounts expressed
from SMN2 has been shown to be partially functional
in mice with severe SMA as it ameliorates the pheno-
type and increases their lifespan from 5 days to 15 days
(Le et al. 2005). Thus, SMN2 is a major modifier of the
disease and SMN2 copy number serves as a prognostic
tool for SMA individuals. The general copy numbers of
SMN2 observed in patients with SMA type I, II, III, and
IV are 2, 3, 3/4, and 4 or higher, respectively. Higher
copy number ([2) of SMN2 has been attributed to gene
conversion of SMN1 to SMN2 (Kubo et al. 2015;
Costa-Roger et al. 2021). Chronic forms of SMA are
36 Page 6 of 17 A Aasdev et al.
associated with gene conversion of SMN1 to SMN2,
resulting in higher SMN2 copy numbers and milder
disease phenotype. Newer methods to detect hybrid
genes and conversions are important to characterize
and to find the other potential disease modifiers. SMN1
or SMN2 partial deletions can also cause SMA. These
partial deletions have been observed commonly for
exons 7 and 8, while deletions covering other exon/
intron regions have also been reported (Gambardella
et al. 1998; Kubo et al. 2015; Stabley et al. 2021). The
SMN1/2 region is populated with a large number of Alu
repeat elements. The most common partial deletion of
6.3 kb which spans SMN1/2 exons 7 and 8 has been
attributed to Alu repeats-mediated recombination, as
they flank the breakpoints (Wirth et al. 1997; Ruhno
et al. 2019).
Parents with one copy of SMN1 are identified as
carriers for the disease. However, cases have been
reported wherein carrier parents had two copies of
SMN1 (Mailman et al. 2001). These carriers are called
silent carriers as they have two copies of SMN1 which
are present on the same chromosome (cis arrangement
of SMN1 copies as opposed to trans, SMN1:2?0) and
null copies on the other one. Six novel SMN1 sequence
variants are identified in the Ashkenazi Jewish popu-
lation that are specific to these duplications and are not
present on single-copy alleles (Luo et al. 2014). Out of
these, two variants, SMN1g.27134T[G and
SMN1g.27706_27707delAT, are tightly linked with the
silent (2?0) carriers. The SNP SMN1g.27134T[G has
been used to identify silent carriers using next-gener-
ation sequencing and qPCR-based approaches (Azad
et al. 2020; Ceylan et al. 2020). These variants have
been observed in higher frequencies in ethnic popula-
tions of Ashkenazi Jews and African Americans (Luo
et al. 2014). The presence of these variants serves as a
useful marker to identify the (2?0) silent carriers
(Alı́as et al. 2018; Ware et al. 2022).
In general, the SMN2 copy number is inversely core-
lated with severity of disease. However, in some cases,
patients with lower copies of SMN2 have been reported
with milder symptoms and those with higher copy
number, with severe phenotypes (Calucho et al. 2018).
The variant c.859G[C in exon 7 of SMN2 creates an
exon splicing enhancer element which significantly
increases the inclusion of exon 7 in SMN2 transcripts and
increases functional SMN protein (Prior et al. 2009;
Vezain et al. 2010). Another variant c.835–34A[G in
intron 6 of SMN2 decreases the binding affinity of a
splicing repressor protein, HuR, and thus increases the
inclusion of exon 7 in SMN2 transcripts (Wu et al. 2017;
Ruhno et al. 2019). Such modifiers result in SMA
discordant families, as siblings with the same number of
SMN2 genes show different clinical presentations.
However, such cases are rare due to the low frequency of
these variants. Several paralogous sequence variants
(PSVs) have been identified between SMN1 and SMN2
genes, but their effect on the disease phenotype remains
to be studied (Blasco-Pérez et al. 2021). Patients with
deletion of one copy of SMN1 and mutations disrupting
the coding region in the other copy are known as com-
pound heterozygotes. Such individuals usually have one
copy of SMN1 but show the SMA phenotype and are
difficult to diagnose with conventional methods. Few
gene modifiers other than SMN2 for intrafamilial varia-
tion of SMA phenotypes are known but their mechanism
of action is not fully understood (Jiang et al. 2019;
Butchbach 2021; Fang et al. 2021).
6. Diagnosis of SMA
The first step of diagnosis of SMA is physical exami-
nation of the proband for symptoms like muscle
weaking or hypotonia, reduced tendon reflexes, loss of
motor functions, and history of motor reflexes, fol-
lowed by counselling for family history for these
symptoms. Occasionally, tests like electromyography
and muscle biopsy are also performed. In some cases,
non-specific biochemical tests are performed to detect
muscle creatine kinase, an enzyme which is released by
deteriorating muscles (Zhang et al. 2012). The most
accurate diagnostic tests for SMA are genetic tests. It is
found that 95% of the SMA patients have homozygous
deletion of exon 7 of the SMN1 gene. Thus, genetic
diagnosis of SMA is usually done by screening for
exon 7. As SMN2 is a major phenotype modifier,
measurement of SMN2 copy number is important for
clinical classification, disease severity, and prognosis of
the disease. The exon7?6 C[T nucleotide change
forms the basis of various molecular assays to detect
the change or quantify SMN1/2 copy numbers. Carrier
screening is important for genetic counselling of ‘at
risk’ families, and in general a priori risk is also pre-
sent even if the family has no previous record of SMA
due to high carrier frequency and de novo cases of
SMA. Family planning and relatives at risk can be
appropriately advised after screening. SMN1 copy
number only forms the basis for carrier identification,
although newer mutations identified associated with
carrier status are also used now (Ware et al. 2022).
Given that SMA is an important health problem, it can
be detected in newborns with high sensitivity, and early
intervention can improve clinical presentation of the
 Indian clinical and research scenarios for SMA
individual. Newborn screening (NBS) for identification
of SMA before the onset of disease symptoms is crucial
to start therapy and supportive care at an early stage.
Thus, rapid and reliable diagnostics are very important.
Many diagnostic assays based on different methods,
including qPCR and multiplex ligation-dependent
probe amplification (MLPA), are available and used to
quantify SMN1/2 copy numbers (Feldkötter et al. 2002;
Anhuf et al. 2003; Arkblad et al. 2006; Fang et al.
2015). qPCR and most other PCR-based methods fall
short as only a few loci can be examined in one reac-
tion. These methods cannot provide information about
gene conversions and novel variants. They are also
inefficient in identifying copy number variations of 4
and above, and require a standard or normal sample run
to quantify SMN1/2 copies. MLPA alleviates some of
these shortcomings as up to about 40 different targets
can be examined in a single reaction. Using MLPA,
deletions vs. conversion of SMN1 can be identified and
SMN1/2 copy numbers of up to 4 can be measured
easily (Alı́as et al. 2011; Vijzelaar et al. 2019). These
factors coupled with ease of use have made MLPA the
gold standard diagnostic technique. Newer methods of
digital PCR (dPCR) are very sensitive and can accu-
rately measure SMN1/2 copy numbers without cali-
bration with a reference sample (Zhong et al. 2011;
Dobnik et al. 2016; Vidal-Folch et al. 2018; Stabley
et al. 2021). Partial deletion and gene conversion can
also be identified. Sequencing, long-range PCR cou-
pled with sequencing, whole-exome sequencing, and
whole-genome sequencing based assays provide addi-
tional information on novel structural variants and
mutations, which, together with clinical data, can pro-
vide information about their role as disease modifier,
important marker, or therapeutic options (Kubo et al.
2015; Ruhno et al. 2019; Chen et al. 2020; Tan et al.
2020; Blasco-Pérez et al. 2021). Higher costs and
specialized personnel/equipment required are the major
drawbacks for next-generation sequencing-based
methods. The diagnostic approaches for SMA are
summarized in figure 3. Recently, simple and non-
invasive saliva-based tests have also been developed
for faster diagnosis of SMA (Wijaya et al. 2021).
Point-of-care testing (POCT) lateral flow-strip-based
assays have also been reported with benefits of high
sensitivity, portability, and cheaper price (Zhang et al.
2021). Large-scale population screening and diagnosis
of patients in inaccessible areas need newer point-of-
care technologies for early diagnosis.
SMA is a highly prevalent disease in India owing to the
large population size and high carrier frequency. Earlier
there were no proper data available on the prevalence of
Page 7 of 17 36
SMA in the Indian population. Very often, due to inad-
equate facilities and lack of awareness among medical
professionals, the patient would die without receiving
clear clinical diagnosis. The lack of expertise in molec-
ular diagnosis in primary and secondary healthcare
centres, ignorance of prenatal diagnosis, psychosocial
issues related to prenatal diagnosis and termination, have
made prenatal diagnosis difficult in India. Kesari et al.
(2005) analysed the carrier status of parents and siblings
of SMA patients and suggested that it is important to
perform carrier status analysis of high-risk individuals to
avoid unwarranted prenatal diagnosis of SMA. SMN1/2
copy number analysis is important for diagnosis of SMA,
as well as carriers, and have been undertaken from
around the start of the century in India. In 2013, Sheth
et al. tested a cohort of 105 Indian patients presenting
hypotonia and lower motor neuron disease, and found
that 65 patients (62%) had SMA, as detected by deletion
of exon 7/ 8 of SMN1/ SMN2 genes by PCR (Sheth et al.
2013). Recent advancements in molecular diagnosis
have resulted in the development of understanding of the
disease prevalence in India. Previously, carrier fre-
quency was estimated to be 1 in 50 for Asian Indians, but
later studies have found it to be higher with 1 in 30–44
(Sugarman et al. 2012; Verma et al. 2020; Nilay et al.
2021; Kaur et al. 2022). Screening of prenatal, new-
borns, and susceptible groups of populations and coun-
selling can invariably help decrease incidence and early
diagnosis can help in better disease management. NBS
programs have been taken up in many developed nations
and are necessary as pre-symptomatic and early symp-
tomatic patients show the best treatment effects (Dan-
gouloff et al. 2021). India also has a desperate need to
start NBS as well as population screening, and screening
individuals with a family history, to curb the burden of
SMA. NBS has been suggested for other diseases as well
in India, and given the severity plus high incidence of
SMA, there is a need to add SMA screening to the
newborn screening panel (Verma et al. 2015).
7. Clinical trials and efficacies of therapies
for SMA
Therapeutic options have been developed for SMA and
are currently in use (figure 4). Major therapeutic options
include (i) gene therapy to deliver the SMN1 gene for
functional SMN protein production, (ii) antisense
oligonucleotide, and (iii) small molecules to modify
SMN2 splicing towards increased inclusion of exon 7 for
the production of full-length functional SMN protein
(Ramdas and Servais 2020). Studies to understand the
36 Page 8 of 17 A Aasdev et al.
Figure 3. Diagnostic approaches for spinal muscular atrophy.
Figure 4. Therapeutic options for spinal muscular atrophy.
long-term effect of these therapeutics are being
undertaken.
7.1 Nusinersen: Antisense oligonucleotide therapy
for SMA
Nusinersen (Spinraza) is an antisense oligonucleotide,
which increases the inclusion of exon 7 in SMN2
mRNA, leading to full-length SMN transcripts and
functional SMN protein. Nusinersen works by blocking
a splice suppressor in intron 7 of SMN2. Nusinersen
was approved by the US Food and Drugs Adminis-
tration (FDA) and the European Medicine Agency
(EMA) in 2016 and 2017, respectively, and was the
first treatment drug for SMA. The drug is given as four
loading doses of intrathecal injections in the first 2
months and then one injection every 4 months (Hage-
nacker et al. 2020). Qiu et al. (2022) have reviewed the
history of the development of nusinersen. The results
 Indian clinical and research scenarios for SMA
of Phase 1 clinical trials for nusinersen were published
in 2016, wherein the safety, tolerability, pharmacoki-
netics, and preliminary clinical efficacy of intrathecal
injections of nusinersen were tested in 28 patients of
type I and II SMA, aged between 2 and 14 years
(Chiriboga et al. 2016). There were no severe adverse
events reported in the study. The results of phase 2
study for the treatment of infantile-onset SMA with
nusinersen were published in 2016 (Finkel et al. 2016).
It was an escalating-dose (6 and 12 mg) study in which
20 participants between the age of 3 weeks and 7
months were intrathecally administered with multiple
intrathecal doses of nusinersen. The results were
encouraging. The final results of the study were
reported in 2021, wherein the efficacy of nusinersen
was monitored over 3 years (Finkel et al. 2021). At the
end of the study, 15 participants were alive, 5 suc-
cumbed most likely due to the progression of the dis-
ease, and 101 serious adverse events were reported in
16 patients. Also, 12 out of 19 evaluable patients had
reached the primary endpoint of an incremental
improvement in the Hammersmith Infant Neurological
Examination section 2 (HINE 2) developmental motor
milestones. In 13 participants with two copies of
SMN2, the HINE 2 motor milestone total score
increased steadily and significantly. The findings
showed favorable safety profile, improved survival,
and achievement of motor milestones over 3 years. The
autopsy results in the study showed that nusinersen was
distributed in the spinal cord as well as brain of the
patients and it could also promote the inclusion of exon
7 in the SMN2 mRNA and promote the production of
functional SMN protein. Later, a sham-controlled,
randomized, double-blind Phase 3 safety and efficacy
trial was performed in 121 SMA infants, randomly
grouped in a 2:1 ratio to undergo intrathecal adminis-
tration of nusinersen (12 mg) or sham control, at 31
centers (Finkel et al. 2017). The doses were adminis-
tered on days 1, 15, 29, and 64 and maintenance doses
were given on days 183 and 302. On days 64, 183, 302,
and 394 (±7 days for each visit), the efficacy endpoints
were monitored. In the interim analysis itself a signif-
icant number of patients who received nusinersen
achieved the motor milestones. Due to the higher
success rate, the trials were terminated earlier. Inter-
estingly, 22% of the infants in the nusinersen group
achieved complete head control, 10% were able to roll
over, 8% were able to independently sit, and 1% were
able to stand; in contrast, none in the control group
were able to achieve these. The survival rate was also
higher in the nusinersen group as compared with the
control group. In another Phase 3 trial, the efficacy of
Page 9 of 17 36
nusinersen was tested for late-onset SMA, with 126
children who had symptom onset after 6 months of age
(Mercuri et al. 2018). Like the infantile-onset SMA
study, this study also showed clearly encouraging
results and ultimately had to be terminated early. There
was a significant increase in the least-squares mean
from baseline to month 15 in the Hammersmith
Functional Motor Scale–Expanded (HFMSE) in the
nusinersen-treated group, which had clinically mean-
ingful improvement in motor function, as compared
with the control group. Recently, Zhong et al. (2023)
reviewed 15 studies that included 967 children, for the
treatment of SMA using nusinersen, and have con-
cluded that there have been rare adverse events asso-
ciated with the treatment and that nusinersen can
effectively reduce the common, serious, and fatal
adverse events in children and adolescents with SMA.
Nusinersen is also shown to be effective in adult SMA
patients (Duong et al. 2021; Fainmesser et al. 2022;
Arslan et al. 2023; Vidovic et al. 2023). The major
drawback of nusinersen is that it needs to be given as
an intrathecal injection in order to circumvent the
blood–brain barrier.
7.2 Onasemnogene abeparvovec: Gene therapy
for SMA
Onasemnogene abeparvovec (Zolgensma) is the gene
therapy option which delivers a functional SMN1 gene.
The delivery is mediated through an adeno-associated
virus serotype 9 (AAV-9) with cytomegalovirus
enhancer and chicken b-actin promoter expressing the
SMN1 gene (Mendell et al. 2017). It was approved by
the FDA in 2019 for children \2 years of age. The
therapy acts systemically to express SMN1, can
potentially cross the blood–brain barrier, and is
administered via a single intravenous injection. A high
viral load of 1.191014 viral genomes/kg body weight
restricts this therapy to children only (Schultz et al.
2019). Intrathecal injections have shown to achieve
higher gene SMN1 expression using lower viral loads
in pigs and is being investigated in human clinical trials
(Federici et al. 2012). The clinical studies START
followed by STR1VE demonstrated the efficacy of
onasemnogene abeparvovec in patients who showed
symptoms of SMA before the treatment.
The START Phase I study was conducted on 15
infants with type I SMA at the Nationwide Children’s
Hospital in Columbus, Ohio, between 5 May 2014 and
15 December 2017. The patients were split into two
groups: group 1 received a low dose of onasemnogene
36 Page 10 of 17 A Aasdev et al.
abeparvovec (one third of that received by group 2) and
group 2 received a higher dose between 1.1 9 1014 and
1.4 9 1014 mg/kg. The results were very encouraging:
100% of the patients in group 2 survived without
breathing support, 11 patients could sit without support
for at least 5s, 9 patients could sit without support for at
least 30s, 11 patients could achieve a ‘Children’s
Hospital of Philadelphia Infant Test of Neuromuscular
Disorders’ (CHOP INTEND) score of more than 40,
and 11 patients could speak and swallow (https://www.
zolgensma.com/clinical-studies/symptomatic-study-
results). The long-term safety and efficacy of
onasemnogene abeparvovec is being monitored in the
START Long Term Follow Up (LTFU) study, in which
the infants who received onasemnogene abeparvovec
in the Phase I START study are being monitored. The
study intends to monitor the safety and efficacy of the
drug in the patients for 15 years. The five-year interim
report of the study, in which the data were analysed
from 21 September 2017 to 11 June 2020 is published
in 2021 (Mendell et al. 2021). The results support the
favorable long-term safety profile of onasemnogene
abeparvovec. A total 13 of the 15 patients, 10 in group
2 (high dose cohort) and 3 in group 1 (low dose cohort)
from the START study, were monitored in the START
LTFU study. Of the 13 patients (3 in group 1 and 4 in
group 2), 7 received a concomitant dose of nusinersen
to maximize health benefits. Of the 13 patients who
received the onasemnogene abeparvovec treatment, 8
showed serious adverse events like acute respiratory
failure, pneumonia, dehydration, respiratory distress,
and bronchiolitis. All 10 patients in group 2 were alive
and did not require permanent ventilation support and
maintained the previously acquired motor milestones.
Two patients, who did not receive concomitant nusin-
ersen, achieved the milestone of standing with assis-
tance. Overall, the study provided evidence for safety
and efficacy of onasemnogene abeparvovec up to 6
years of age. More recently, the updates of these
patients were reported, and the therapy continued to
demonstrate the favorable risk benefit profile and dur-
able efficacy up to 8 years post the dose (Mendell et al.
2023).
STR1VE is an open-label, single-arm, multicenter
Phase III clinical trial involving 22 symptomatic type 1
SMA patients that received onasemnogene abepar-
vovec (Day et al. 2021). The patients received the
therapeutic dose of onasemnogene abeparvovec at an
average age of 3.7 months (0.5–5.9 months) and were
monitored through 18 months of age. Of the 22
patients, 13 could sit independently for at least 30 s (as
compared with 0 of 23 untreated patients in the cohort),
and 20 were alive without permanent ventilation sup-
port. All patients had at least one adverse event, most
common being pyrexia. The serious adverse events
included bronchiolitis, pneumonia, respiratory distress,
and respiratory syncytial virus bronchiolitis, and three
serious adverse events were possibly related to the
treatment. Overall, the safety and efficacy results of the
STR1VE study in the US and Europe were concurrent
to that of the START study (Mercuri et al.
2019a, b, 2021). Similar encouraging results were also
reported by another study involving a cohort of 25
children in Dubai (Chencheri et al. 2023).
SPR1NT is a single-arm, multi-center Phase 3
clinical trial study to investigate the safety and effi-
cacy of onasemnogene abeparvovec in 14 pre-
symptomatic infants of less than 6 months of age
having biallelic SMA-associated mutations in the
SMN1 gene and two copies of the SMN2 gene and
are expected to develop type 1 SMA (Strauss et al.
2022). All 14 children treated with onasemnogene
abeparvovec survived without permanent ventilation
at 14 months and were able to sit independently for
at least 30 s during any visit before 18 months. No
adverse events related to the treatment were reported
and the treatment was effective and well tolerated by
the pre-symptomatic children.
For adult SMA patients, onasemnogene abeparvovec
has not been tested due to the higher viral titres, AAV
exposure with age, antibodies thereof, and complica-
tion of using the AAV vector. Issues, such as immune
response against AAV-9 capsid proteins, neuronal and
hepatotoxicity, question the use of the AAV-9 delivery
system at higher doses in adults (Ertl 2022). A recent
study found that the antibodies for AAV in adult SMA
patients were not elevated and paves the way for a
treatment for adult SMA patients (Stolte et al. 2022).
Although overall, gene therapy showed promising
results, there were two deaths reported in children
administered with Zolgensma, as reported by Novartis,
due to liver function failure, a side effect of the AAV
vector used (https://www.statnews.com/pharmalot/2022/
08/11/novartis-zolgensma-liver-failure-gene-therapy-
death/). Liver failure was already included in the
drug information, but will be looked at more closely
hereafter.
7.3 Risdiplam: Small molecule-splicing modifier
therapy for SMA
Risdiplam (Evrysdi) is a small molecule which modi-
fies SMN2 splicing to increase exon 7 in mRNA to
 Indian clinical and research scenarios for SMA
produce full-length SMN transcripts (Naryshkin et al.
2014). It was approved by the FDA in August 2020 for
the treatment of SMA for patients of[2 months of age.
The drug is taken as an oral medication daily, can cross
the blood–brain barrier, and has the benefit of systemic
distribution (Poirier et al. 2018). A two-part open-
labelled phase 2–3 study to evaluate the safety and
efficacy of risdiplam in type 1 SMA infants between
the age of 1 and 7 months demonstrated the potential of
using the drug for SMA. Part 1 of the study mainly
involved monitoring of safety, pharmacokinetics,
pharmacodynamics, analysis of blood SMN protein
concentration, and determination of the dose for part 2
of the study (Baranello et al. 2021). A total of 21
infants were enrolled in the study, and 4 of them
received a low dose of 0.08 mg of risdiplam per kilo-
gram of body weight per day and 17 received a high
dose of 0.2 mg per kilogram per day at 12 months. A
dose-dependent increase in the concentration of SMN
protein in the blood was observed in these patients after
receiving the drug. A total of 24 serious adverse events
including pneumonia, respiratory tract infection, res-
piratory distress, and acute respiratory failure was
observed. Four infants died of respiratory complica-
tions. A dose of 0.2 mg per kilogram per day was
selected for the part 2 of the study (FIREFISH part 1)
which involved 41 participants from 14 centers in 10
countries (Darras et al. 2021). Of the 41 children, 12
were able to sit without support for at least 5s after 12
months of treatment, 23 had a CHOP-INTEND score
of 40 or above, with 37 having an increase of at least 4
in the CHOP-INTEND score from the baseline, 32 had
achieved a HINE-2 motor milestone response, and 48
serious adverse events were reported, the most com-
mon being pneumonia, bronchiolitis, hypotonia, and
respiratory failure. The FIREFISH part 2 study
involved the monitoring of the 41 patients over 24
months treatment (Masson et al. 2022). Of the 38
patients who 30s continued the study, 18 were able to
sit for at least 30s without support, and none could
stand or walk alone after 24 months of treatment. Also,
22 patients reported upper respiratory tract infection as
the most common adverse event, 16 infants reported
pneumonia as the most common serious adverse event,
and 3 infants reported respiratory distress. FIREFISH is
an ongoing trial to monitor the safety and efficacy of
risdiplam. At 36 month, 84% patients were alive, did
not need permanent ventilation, and maintained or
improved the motor skills and developmental mile-
stones (Darras et al. 2023).
SUNFISH is a randomized, double-blind, placebo-
controlled Phase 3 clinical trial study to test the safety
Page 11 of 17 36
and efficacy of risdiplam in patients with type 2 or non-
ambulant type 3 SMA of a broad age group of 2–25
years. The first-year interim results of the study
demonstrated that the drug was well tolerated in patients
and led to increase in SMN protein levels in the blood
(Mercuri et al. 2019a, b). The SUNFISH part 1 (12
months report) and part 2 (24 months report) study
involved 180 patients from 42 hospitals, randomly
grouped into two groups in the ratio of 2:1; 120 received
risdiplam and 60 received a placebo. The study reported
a significant improvement in motor function in the group
treated with risdiplam as compared with the placebo-
treated group, with similar incidence of serious adverse
events among both the groups at 12 months and 24
months of treatment (Mercuri et al. 2022; Oskoui et al.
2023). The RAINBOWFISH trail for genetically diag-
nosed, but asymptomatic/pre-symptomatic individuals
of age 6 weeks or below is ongoing, with interim results
showing all patients free of ventilation and no adverse
effects (Finkel et al. 2022).
Clinical trials to test the effect of the one drug over
the other are also in progress. Novartis started an open-
label multi-center study, named STRENGTH, to test
the efficacy of intrathecal SMA gene therapy on
patients who discontinued treatment with nusinersen or
risdiplam, and is recruiting participants (https://www.
novartis.com/clinicaltrials/study/nct05386680). Biogen
started the RESPOND trial to study if patients who
previously took a dose of gene therapy could benefit
with the further use of nusinersen (https://www.
biogentriallink.com/en-us/home/find-a-clinical-trial/
clinical-trials-study-details.html?nctId=NCT04488133).
In yet another study named JEWELFISH, the safety of
risdiplam on patients previously treated with any SMA
drug was evaluated and no significant difference from
the previous safety profiles of risdiplam was observed
with maintained SMN protein levels (Chiriboga et al.
2019, 2023).
7.4 Plausibility of mRNA therapy for SMA
The remarkable success of mRNA vaccines against
severe acute respiratory syndrome coronavirus-2
(SARS-CoV-2) has rejuvenated interest in using
mRNA encoding the protein of interest as a therapeutic
entity against rare genetic disorders involving loss of
functional proteins. In mRNA therapy, mRNA encod-
ing the protein of interest, encapsulated in carriers like
lipid nanoparticles (LNPs) or cellular membranes, is
injected into the target cell or tissue. The injected
mRNA produces the functional protein using cellular
36 Page 12 of 17 A Aasdev et al.
machinery and thus rescues protein functionality and
ameliorates disease symptoms. Earlier, studies involv-
ing mRNA therapy that have yielded some encourag-
ing results include the use of mRNA-encoding vascular
endothelial growth factor (VEGF) for heart failure and
mRNA-encoding clustered regularly interspaced short
palindromic repeats (CRISPR) and CRISPR-associated
protein-9 (Cas-9) for hereditary amyloidosis (Collén
et al. 2022; Gan et al. 2019; Intellia and Regeneron
2022; Rohner et al. 2022). As the mechanism under-
lying the pathogenesis of SMA involves loss of pro-
duction or inadequate synthesis of the functional SMN
protein, mRNA therapy is a promising option for SMA.
The potential of use of SMN1 mRNA encoding the full-
length SMN protein, under appropriate regulatory ele-
ments, packaged in LNPs can be explored. As LNPs do
not cross the blood–brain barrier efficiently, more
research is required for the development of mRNA
therapy for SMA. The research required would include
optimizations with respect to mRNA delivery system,
target tropism, sustained expression, and chronic dos-
ing. Targeting solutions which can deliver the thera-
peutic mRNA efficiently to both the central nervous
system as well as the peripheral nervous system would
be crucial in this context.
All three available therapies for SMA have shown to
be safe and tolerable in patients, the SMA phenotype
was improved, patients gained motor milestones, res-
piratory function was ameliorated, and there was no
requirement for ventilator support. The long-term out-
comes of these treatments are still under investigation.
The treatment has shown to be more effective in infants
when started early (Eggermann et al. 2020). Therefore,
NBS and population screening are crucial to identify
and start treatment at an early stage. Biomarkers other
than SMN2 are needed for validation of therapy and
predicting the course of the disease phenotype
(Schorling et al. 2020; Wirth et al. 2020). Other
treatment options to alleviate disease symptoms include
improving motor neuron function, muscle function
enhancement, and promotion of cell growth (Keinath
et al. 2021).
Although, globally there are hundreds of ongoing
clinical trials to test the safety and efficacy of the three
drugs, nusinersen, onasemnogene abeparvovec, and
risdiplam, for the different types of SMA, the data
pertaining to Indian patients are not available. Recently,
Novartis Pharmaceuticals has proposed a multi-center,
randomized, sham-controlled, double-blind Phase 3
clinical trial study (NCT05089656) to investigate the
safety, tolerability, and efficacy of IT OAV101 in sitting
and non-ambulatory SMA participants, which involves
patient recruitment from India. The patient recruitment
is ongoing through three Indian centers: PD Hinduja
National Hospital and Medical Research Centre,
Mumbai; All India Institute of Medical Sciences
(AIIMS), New Delhi; and Sir Ganga Ram Hospital,
New Delhi. Another randomized placebo-control study
to test the efficacy of valproate, a histone deacetylase
(HDAC) inhibitor, and levocarnitine, on children with
SMA is being conducted in India (NCT01671384) at
AIIMS, New Delhi. Hopefully, in the near future, the
results from these studies would be available so as to
understand drug efficacies in the Indian context.
8. Conclusion
With a carrier frequency of 1:38–44, SMA is not that rare
in India. Carrier status analysis of couples with or without
family history, genetic counselling, and incorporation of
SMA diagnosis in newborn screening can help reduce the
disease burden. Indian patients have limited access to
therapeutic options, due to exorbitant costs. There is a
dire need for relaxing the patent laws associated with
these drugs and promoting their indigenous production.
Figures
Figures are made using BioRender.
Acknowledgements
The authors thank Dr. Rakesh K Mishra and Dr. Alok
Bhattacharya for their directions and insightful inputs.
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