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Antisense Mediated Induction of SMN Expression in Brain and Spinal Cord Tissue
Improves Bodyweight and Righting Response in the SMA Mouse
Jason H
1
Williams ,
Rebecca C Schray, Xiangying Guan, Carlyn A Patterson, Melanie K Tallent, Gordon J Lutz.
Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, PA
1Current Affiliation - Laboratory for Applied PK/PD, Division of Clinical Pharmacology and Therapeutics, Children’s Hospital of Philadelphia Philadelphia, PA
II
2nd-3rd
7-18 months sit, but never stand
III
<30 years
decade
may ambulate independently normal lifespan
• Proximal SMA (types I,II,III) has been linked to the homozygous
deletion of the SMN1 gene on human chromosome number 5. SMN1
deletion results in depletion of SMN protein in every cell in the body
• Only spinal motor neurons are affected by loss of SMN.
Clinical relevance of Antisense strategy
Injection
site
100
Lateral
ventricles
SMA type II
90
SMA type III
80
C
D
Olfactory
ventricle
70
4th ventricle
60
Left-bottom Cross sections from SMA mice
injected with AO-FAM showed distribution
outside of ventricular regions (D-F) and under
high magnification AO can be seen as punctuate
structures (G-I). Three time-points are shown
including mice injected once on P1 and analyzed
24 h later (D, G), mice injected on P1 and P3 and
analyzed 24 h later (E,H) and mice injected on P1,
3, 5, 7, 10 and analyzed 48 h later (F, I). Brain
sections from non-injected SMA mice are shown
as a control (A-C).
50
40
30
20
10
0
1
2
3
4
5
SMN2 copy number
Molecular Genetics of SMN
• SMN2 is nearly identical to SMN1 but predominantly aberrantly
spliced resulting in decreased amounts of functional SMN.
SMN1
6
7
Left -top ICV injection of Trypan blue dye (A,C)
and FAM-AO (B,D) show distinct distribution of
AO throughout lateral, olfactory and the 4th
ventricle. Dorsal (A, B) and Ventral (C, D) views
are shown.
8
(A)n
100%
6 7 8 (A)n
~90%
68
(A)n
SMN FL
Δ7
(100) (15) (22)
Discovery of an effective Antisense Oligonucleotide
• Antisense Oligonucleotide (AO) mediated steric-blocking of an
intronic splicing element within intron 7 of the SMN2 pre-mRNA
was shown to correct aberrant-splicing and produce nearly 100%
full length SMN in SMA patient fibroblasts (Singh et al., 2006).
B
SMA wt
(22) (31)
(25)
Cervical
SMA injected
30
Tubulin
FL
SMN Δ7
(25) (100) (22) (19) (27)
C
wt SMA
OBJECTIVES
(19) (27)
(22) (31) (25)
Lumbar
SMA injected
Tubulin
FL
• Determine whether post-natal induction of SMN expression in
brain and spinal cord tissue improves weight and motor function of
SMA mice at postnatal day 12.
25
*
SMA control
SMA PEG-PEI-AO
SMA control
(28)
(27)
(26)
Cervical
SMA injected
SMA
FL
SMN Δ7
(100) (15)
**
A
30
20
12
10
Tubulin
SMA wt
8
(51)
(41)
AO alone
SMN FL
Lumbar SC
(61) (79)
non-SMA
10
0
(51)
Lumbar
SMA injected
C
Hippocampus Cervical SC
(41)
Tubulin
SMA AO
*
SMA
(41)
Δ7
PEG-PEI-AO
(15)
(100) (33)
(28)
(43)
(29) (25)
SMA
Left SMN expression is significantly increased in hippocampus and spinal cord
6
regions of AO treated SMA mice when measured as a percentage of wild-type
controls (N=5 for treated and control, *P<0.001, **P<0.01).
4
A
Bodyweight
analysis of SMA2 mice from birth-P12
12
0
non-SMA
10
AO alone
Left Weights of Normal(▲), SMA (■),
10
9
8
7PEG-PEI-AO
6
5 SMA
4 and
3 (○),
2 AO
1 SMA
(■) from P1-P12.Postnatal Day
B
PEG-PEI-AO
8
*
SMA
6
6
4
**
P9
at P9
weight at
weight
**
**
5
4
(100) (25) (17) (20) (21) (36) (26)
(11)
2
3
4
5
6
7
Postnatal Day
8
9
10
11
12
3
SMA
AO
PEG-PEI-AO
B Right Increase in SMA AO treated body weight relative to SMA controls on days 9
*
15
10
5
Hippocampus Cervical SC
Lumbar SC
Right SMN expression is increased in cervical spinal cord regions of PEG-PEI-AO treated
SMA mice when measured as a percentage of wild-type controls (*P<0.05; N=7 for PEGPEI-AO, N=5 for SMA control).
Overview of Western blot
• 20 µg total protein was run on pre-cast 4-15% gradient SDS-PAGE gels (Bio-Rad).
• Primary antibodies for SMN(38 kDa) and Tubulin (50 kDa) protein were mouse
monoclonal anti-SMN (1:2000, BD Biosciences) and anti-tubulin (1:30,000, Abcam).
• Donkey anti-mouse IRDye 800CW secondary antibody (Li-COR Biosciences) was
applied for 1h and membranes were scanned and quantified on an Odyssey Infrared
Imaging System with Odyssey v2.1 software (Li-COR Biosciences).
11
12
1
2
3
4
5
6
7
8
9
SMA
AO
1
2
3
4
5
Left
(sec)
30
30
30
30
5
4
30
2
30
4
2
3
4
30
Right
(sec)
30
30
30
30
30
8
30
30
30
5
30
3
30
1
Best
(sec)
30
30
30
30
5
4
30
2
30
Yes/No
(1/0)
0
0
0
0
1
1
0
1
0
4
2
3
4
1
1
1
1
1
1
Left Chart of best
righting times from
3 trials per side for
SMA and SMA AO
treated mice.
8
7
Best side Righting time
6
5
SMA control
4
SMA AO
3
2
1
0
Right Best righting times of
SMA and SMA AO treated mice
when placed on either side.
0-4
5-10
10-30
>30
Seconds
P12
at P12
weight at
weight
weightand
weight
at
at P9
P9SMA PEG-PEI-AO treated showed a significant on
(*, P<0.01) and 12 (**P<0.05)
6
weight
weight
at
at P12
P12
day 9 (**P<0.05 ) but not day
12.
N=5 for AO and SMA control, N=7 for PEG-PEIAO treated.
**
5
Full-length SMN2 measured by real-time PCR
• There4 was a 1.4-fold increase in full-length SMN2 transcripts in
SMA AO-treated compared to SMA control.
20
0
SMN Δ7
• Deliver AO to brain and spinal cord tissue of SMA mice and
quantify SMN expression by Western blot.
Left SMN expression shown for
hippocampus (A) brain, cervical (B) and
lumbar (C) spinal cord extracts (N=7). FLSMN and Δ7-SMN are indicated by arrows.
SMN expression (%)
(A)n
wt
*
40
1
Tubulin
8
(18)
0
SMA injected
wt SMA
~10%
7
(100)
Quantification of SMN expression by Western Blot
A
6
Δ7
2
Hippocampus
SMN2
SMN FL
B
50
B
SMA type I
Percent of patients(%)
• The range of severity has
been inversely correlated
with the SMN2 gene copy
number (Wirth et al.).
• The occurrence of multiple
SMN2 copies makes
Antisense mediated splice
correction a promising
potential pharmacotherapy.
RESULTS
Intracerebroventricular injection of fluorescent
Antisense Oligonucleotides
A
Tubulin
70
60
• Individual mice were placed on a flat surface and both right
and left side “righting” ability was measured. The number
of seconds to right is listed in the chart. When >30 seconds
was needed to right, a score of 30 was recorded.
• There was a significant (p = 0.035) difference when righting
was measured binomially (yes/no) and analyzed whether
AO-treatment improved righting (Fisher’s exact, one-sided).
SMA injected
wt SMA
Weight (g)
Progression
death by age 2
Test of motor function shows improvement in AO
treated SMA mice
Hippocampus
A
Weight (g)
Subtype Age at onset Clinical Features
I
0-6 months never sit
Right AO treated SMA mice showed
increased SMN expression in
hippocampus (A) brain extracts and
cervical (B) and lumbar (C) spinal cord
extracts (N=5). FL-SMN and Δ7-SMN
are indicated by arrows.
Weight (g)
Table 1. Sub classification of SMA type
• SMNΔ7+/+SMN2+/+Smn-/- mice were used as the model for SMA.
Mice live for ~14 days and are severely functionally compromised
exhibiting decreased weight and motor function measured by ability
to right themselves when placed on their side (Le et al., 2005).
• 1μl of 2’-O-Methyl, phosphorothioated AO (5’-AUUCACUUUCAU
AAUGCUGG-3’, Trilink Biotech, San Diego, CA) was injected at a
concentration of 1 µg/µl either diluted in sterile saline or complexed
with a characterized Polyethylene glycol-Poly (ethylene imine)
(PEG-PEI) nanopolymer (Williams et al., 2006, 2008).
• Cerebrospinal fluid injection was performed into the left and right
lateral ventricle on post-natal day 1, 3, 5, 7 and 10.
• Functional analysis and tissues harvest was done on day 12.
Weight (g)
• Proximal recessive Spinal Muscular Atrophy (SMA) is the leading
inherited cause of infant mortality with a prevalence of 1:10,000 and
carrier frequency of 1:50.
• Three subtypes distinguish the range of severity and are categorized
based on age at onset and highest motor function achieved.
RESULTS
RESULTS
Improved SMN expression in Brain and Spinal Cord:
AO-saline is more effective than Nanopolymer-AO
Number of Mice
METHODS
SMN expression (%)
BACKGROUND
Overview of real-time PCR
3
• TaqMan primers
for detecting full-length SMN2 were previously
SMA and probes
AO
PEG-PEI-AO
characterized for detecting FL-SMN2 in human samples (Sumner et al 2006 Neurology)
and were used to detect the amount of human SMN2 mRNA in SMA mice.
• HPRT1 control primers (Applied Biosystems) were used as a control.
• SMN2 or HPRT1 transcript levels were quantified by the threshold cycle (Ct) method
using independent threshold settings.
ACKNOWLEDGEMENTS
• This work was supported by the Spinal Muscular Atrophy Foundation
• We thank: Gideon Dreyfuss (University of Pennsylvania, HHMI) for technical support
regarding quantitative Western blotting of SMN
CONCLUSIONS
• Overall, this study illustrates for the first time that AOs which
rectify aberrant splicing of SMN2 are effective in vivo for
induction of SMN expression in an SMA mouse model and are
promising compounds for treatment of SMA.
• Studies are ongoing to evaluate the dose-response relationship
and the extent to which AO treatment increases lifespan in
SMA mice.
• Novel nano-carriers designed to deliver AO across the blood
brain barrier will need to be designed for AO therapy in SMA
patients.
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