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Page 16 of 35 Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01

life expectancy but develop muscle weakness with time. Homozygous deletions or deleterious mutations in
the SMN1 gene are present in all SMA patients.

SMN2 gene is a paralog of SMN1 which results from the duplication of the 5q13 region gene and has been
identified in a more centromeric position. The SMN2 gene is unique to Homo Sapiens and differs from
SMN1 in less than 20 nucleotides, but a functionally crucial difference is the existence of thymine instead of
[94]
cytosine in exon 7 . This modification inactivates a splicing enhancer and, on the opposite, creates a
splicing silencer. Consequently, exon7 is skipped by the splicing machinery in about 90% of maturated
SMN2 mRNA, leading to an unstable non-functional shorter SMN2 protein [Figure 9]. Still, about 10% of
the maturated SMN2 transcripts contain Exon 7 and lead to a functional SMN2 protein with identical
neuroprotective properties as those of SMN1.

The SMN2 gene is located in an unstable chromosomal region and is consequently present under a variable
copy number in the population, leading to variable production of functional SMN2 protein. The broad
spectrum of the severity of the SMA disease has been correlated with the number of SMN2 copies, which is
coherent with the assumption that more SMN2 copies might, at least partially, compensate for the absence
of SMN1 protein. As schematized in Figure 9, the presence of multiple copies of the SMN2 gene partially
alleviates the disease symptoms by allowing more functional SMN2 protein to be produced, with Type I
patients generally possessing 2 SMN2 copies, Type II having 3 SMN2 copies, and Type III having 3-4 SMN2
gene copies. Thus, the number of SMN2 copies is clearly inversely correlated to SMA severity, but other
genes have also been proposed to be involved in SMA severity [95,96] .

The observation that SMN2 was a strong disease modifier established the basis for the quest to increase the
production of a complete and functional SMN2 protein by antagonizing the exon 7 skipping reaction. Using
a systematic study of minigene mutants with different deletions at the 5’ end of intron 7, a novel inhibitory
element located immediately downstream of the 5' splice site in intron 7 was identified, which was called
[97]
intronic splicing silencer N1 (ISS-N1) . A pragmatic approach consisting of screening a large number of
overlapping ASOs targeting introns 6 and 7, as well as exon 7, identified several sites on the 3 sequences
whose steric blocking would inhibit undesired exon 7 skipping and confirmed that ISS-N1 was the most
[98]
promising target sequence for enforcing full SMN2 protein expression . Thus, ASOs targeting a sequence
at approximately 10 nucleotides downstream of the 5’splice site were further developed, leading to the drug
Nusinersen.

Nusinersen (Spinraza ) was made available in 2016 as the first treatment for spinal amyotrophy.
TM
Remarkably, Nursinersen’s discovery rationale was based on the above-described thorough elucidation of
the molecular mechanism of SMN1 and SMN2 mRNA maturation. Nusinersen increases child survival and
is administered by repeated intrathecal delivery (i.e., in the spinal cord). Nusinersen is an 18-nucleotide
oligomer whose 5’-3’ sequence is: UCACUUUCAUAAUGCUGG. It is a highly modified ASO having a full
phosphorothioate backbone and 2’O-Me modified ribose to increase metabolic resistance and
bioavailability. In addition, all pyrimidines are 5-methylated: uracil replaced by thymine, and cytosine
replaced by 5’-methyl cytidyl. The Nusinersen IUPAC formula is detailed in Table 2.

Nusinersen displays high efficacy for promoting exon 7 recovery into SMN2 protein. It was shown that
increasing SMN exclusively in peripheral tissues completely rescued muscle necrosis in mild SMA mice
models and robustly extended survival in severe SMA mice, with significant improvements in vulnerable
tissues and motor function . However, CNS effects upon IV injection could only be observed in neonates
[99]
but not in adult mice. It is known that the blood-brain barrier is permeant in neonates and is subsequently