Gene Therapy in Spinal Muscular Atrophy (SMA) Models Using Intracerebroventricular Injection into Neonatal Mice - Gene Delivery and Therapy for Neurological Disorders - page 301

a

U2AF

70K

U2
snRNP

U1
snRNP hnRNP-G SRp30c

SF2/ASF hTra2b1 hnRNP-Q

Element 1 ISS-N1

C
T

Exon 6 Exon 7 Exon 8

PTB/Fuse

hnRNPA1/2

hnRNPA1

Decrease Exon7 Inclusion Increase Exon7 Inclusion

b

SMN2 pre mRNA

Cis-splicing

90%

10% Exon 6 Exon 7 10%

Exon 6 Exon 8

90% P(PY)

BP

Exon 6 Exon 7 Exon 8

7-11

BP

7 -11

M13

U6 tsRNA + 7-11

Poly A

P(PY)

Trans-splicing

SMN1 Exon7

M13 Poly A

Trans-spliced RNA

Exon 6 Exon 7

Fig. 1 Schematic of the exon 7 regulation within SMN pre-mRNA and trans -splicing strategy to replace the
SMN2 exon 7. ( a ) SMN pre-mRNA is regulated by cis elements (within intron 6, exon 7, and intron 7) and splic-
ing factors which are involved in the inclusion or exclusion of exon 7. The components of the splicing machin-
ery are shown in yellow . The positively acting splicing factors are shown in green . The negatively acting
sequences and splicing factors are shown in red . The C-T transition that creates a splice silencer site in SMN2
transcript is indicated. ( b ) Cis -splicing of SMN2 pre-mRNA produces two mRNAs due to an alternative splicing
event. Trans - splicing strategy utilizes a tsRNA that consists of (1) an antisense domain that binds to intron 6
and blocks the endogenous branch point; (2) the splicing domain with optimal branch point, polypyrimidine
tract, and 3' splice site that carry out the splicing reaction between the SMN2 pre-mRNA and tsRNA; and (3)
the SMN1 exon 7 which replaces SMN2 exon 7 following a trans -splicing event. The generation of the trans -
spliced RNA is verifi ed by using a primer specifi c for the M13 sequence located downstream of the exon 7 stop
codon. Antisense oligonucleotide (7-11) expressed from the U6 promoter is located downstream of the tsRNA
sequence and binds the 3 ′ splice site of exon 8

subcutaneous injections [ 46 ]. Gene therapy applications utilizing
gene correction system by therapeutic RNAs in SMA have been
reviewed in detail [ 50 - 54 ]; therefore, we will only discuss the gene
replacement therapy using viral vectors.

First gene replacement strategy in SMA utilized a lentiviral vector
expressing full-length human SMN cDNA that was retrogradely
transported into motor neurons following multiple injections
into a large number of muscles in SMN

1.1.5 SMA Gene
Replacement Therapy

7 mice [ 55 ]. The out-
come was more viable motor neurons and a modest extension in

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