Gene Therapy in Spinal Muscular Atrophy (SMA) Models Using Intracerebroventricular Injection into Neonatal Mice - Gene Delivery and Therapy for Neurological Disorders - page 299

relevant to intrathecal injection in patients, opens the door to
understand these potential impediments in large animals and con-
sequently in SMA patients. In this chapter, we will review the most
recent gene therapy applications in SMA animal models and dis-
cuss their results. We will also demonstrate the outcome of our
gene therapy applications using single-stranded AAV2 (ssAAV2)
and scAAV9 in SMA mice. Lastly, we provide a detailed protocol of
the CNS delivery using ICV injection and illustrate the sequential
steps of injection in neonatal mice.

1.1 Molecular
Genetics of SMA

SMA is an autosomal recessive disorder that is the leading genetic
cause of infantile death. SMA is the most common inherited motor
neuron disease and occurs in approximately 1:6,000 live births
[ 25 , 26 ]. Infants with the most severe form of SMA (type I) have
normal strength at birth but exhibit progressive weakness within a
few months. Death due to respiratory failure usually ensues within
1-2 years [ 26 ]. Types 2 and 3 SMA present with weakness during
childhood and usually exhibit variable degrees of physical impair-
ment [ 27 ]. These observations imply that motor neuron dysfunc-
tion develops after a latent period of a few months to years and,
importantly, suggests that a temporal window for therapeutic
intervention exists during these presymptomatic stages.

The genetic basis of SMA is the loss of SMN1 located on chromo-
some 5q [ 20 ]. Interestingly, a human-specifi c copy gene is present
on the same region of chromosome 5q called SMN2 [ 20 ]. SMN2 is
nearly identical to SMN1 ; however, mutations in SMN2 have no
clinical consequence if one intact copy of SMN1 is retained. The
reason why SMN2 cannot prevent disease development in the
absence of SMN1 is that 90 % of SMN2 -derived transcripts are alter-
natively spliced giving rise to a truncated and unstable protein
(SMN

1.1.1 SMN Genes

7) that lacks the 16 amino acids encoded by SMN exon 7
(normally the last coding exon) [ 28 , 29 ]. A single non-polymorphic
nucleotide difference (C6T) between SMN1 and SMN2 is respon-
sible for the alternative splicing of the SMN2 transcripts [ 29 , 30 ].
Importantly, the C/T transition does not alter the protein coding
capacity of SMN2 , and therefore, full-length transcript derived from
SMN2 generates an identical protein to SMN1 . Complete loss of
SMN protein is embryonic lethal, and thus, SMA phenotype is
developed as a result of 10 % of functional SMN protein generated
by SMN2 gene. On the other hand, SMN2 is a critical genetic mod-
ifi er due to two major reasons: (1) more copies of SMN2 alleviate
the SMA phenotype, and (2) SMN2 splicing correction is one of the
major therapeutic interventions in SMA gene therapy.

ʔ

SMN is a 294 amino acid (38 kDa) protein that is enriched in a
variety of tissues in the CNS including motor neurons in the spinal
cord and appears to be involved in a large number of RNA

1.1.2 SMN Protein
and Function