Biomedical Engineering Reference
In-Depth Information
the virus construction, the purity/quality of the virus, and even
variations in the injection methods. According to our study, a low
dose (2 × 10
10
genomic copies) of scAAV9-SMN is still suffi cient to
rescue the SMA mice effi ciently when delivered through ICV
rather than IV. Therefore, the dose of the virus depends greatly on
other parameters of the gene therapy to reach a high level of effi -
ciency. In conclusion, appropriate combination of all the parame-
ters of the gene therapy mentioned above is critical to achieve a
suffi cient level of rescue.
1.2 Summary of Our
Research in SMA Gene
Therapy
Splicing of
SMN
exon 7 is regulated by many
cis
- and
trans-
acting
factors that function positively or negatively leading to either inclu-
sion or exclusion of exon 7 (Fig.
1a
) [
33
,
50
]. Our efforts in SMA
gene therapy include correction of
SMN2
pre-mRNA splicing
(increasing the inclusion of exon 7) by direct delivery of the thera-
peutic RNAs (bifunctional and
trans
-splicing) as well as modifi ed
oligonucleotides into the CNS by ICV injection [
44
,
80
-
83
]. We
have also utilized ssAAV2 to deliver the therapeutic RNAs and
scAAV9-SMN to carry out gene replacement therapy [
23
,
24
,
75
].
Our research involving the therapeutic RNAs (in the form of plas-
mid DNA and modifi ed oligonucleotides) has been reviewed in
detail previously [
50
,
53
]. Here, we only describe the outcome of
our gene therapy research utilizing viral vectors.
Our fi rst attempts to exploit viral vectors involved the construction
of traditional ssAAV2 expressing therapeutic RNAs (bifunctional
and
trans
-splicing) which showed great effi cacy in increasing the
SMN levels in cell-based assays [
84
,
85
]. Our initial
trans
-splicing
RNA (tsRNA) molecule was designed to target intron 6 within the
SMN2
pre-mRNA and replace the following 3' exon (exon 7) with
SMN1
exon 7 [
85
]. This construct (designated pM13) consisted of
the following: an SMN intron 6 annealing sequence approximately
130 bp, an optimized heterologous splice site, the
SMN1
exon 7
sequence, and a specifi c sequence (M13 primer) immediately
downstream of the native stop codon at the end of exon 7 to allow
straightforward detection of the
trans
-spliced product by RT-PCR
(Fig.
1b
) [
85
]. However, the effi ciency of the viral vector (ssAAV2)
expressing this
trans
-splicing RNA was negligible in SMA mouse
models. Therefore, we optimized the tsRNA by adding an anti-
sense (7-11) to block splicing at the downstream exon (exon 8) to
create more opportunities for a
trans
-splicing event (designated
pMU3) (Figs.
1b
and
2a
) [
81
,
86
]. We concentrated ssAAV2 con-
taining pMU3 by cesium-chloride gradient centrifugation and
delivered 1 × 10
11
genomic copies of the vector through ICV injec-
tion into SMN
1.2.1 Development
of ssAAV2 Vectors
7 mice at P2. Our goal was to analyze the func-
tionality of the vector to generate a
trans-
spliced RNA from the
SMN2
pre-mRNA in the CNS of the injected SMN
ʔ
7 mice. Using
this approach, the 400 bp
trans
-spliced RNA was detected in the
ʔ
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