Religious background Economic background Political background.
BACKGROUND
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SM A Weight(g) AO P EG -PEI-A O SM A Weight(g) AO P EG -PEI-A O * SM A Weight(g) AO P EG -PEI-A O 3 4 5 6 weightatP 9 weightatP12 SM A Weight(g) AO P EG -PEI-A O * ** ** BACKGROUND CONCLUSIONS REFERENCES Antisense Mediated Induction of SMN Expression in Brain and Spinal Cord Tissue Improves Bodyweight and Righting Response in the SMA Mouse Jason H Williams 1 , 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 1 Current Affiliation - Laboratory for Applied PK/PD, Division of Clinical Pharmacology and Therapeutics, Children’s Hospital of Philadelphia Philadelphia, PA Le TT, Pham LT, Butchbach ME, Zhang HL, Monani UR, Coovert DD, Gavrilina TO, Xing L, Bassell GJ, Burghes AH (2005) SMNDelta7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN. Hum.Mol.Genet. 14: 845-857 Singh NK, Singh NN, Androphy EJ, Singh RN (2006) Splicing of a critical exon of human Survival Motor Neuron is regulated by a unique silencer element located in the last intron. Mol.Cell Biol. 26: 1333-1346 Sumner CJ, Kolb SJ, Harmison GG, Jeffries NO, Schadt K, Finkel RS, Dreyfuss G, Fischbeck KH (2006) SMN mRNA and protein levels in peripheral blood: biomarkers for SMA clinical trials. Neurology 66: 1067-73 Williams JH, Schray RC, Sirsi SR, Lutz GJ. Nanopolymers improve delivery of exon skipping oligonucleotides and concomitant dystrophin expression in skeletal muscle of mdx mice. BMC Biotechnology 2008 Apr 2;8(1):35. Williams JH, Sirsi SR, Latta DR, Lutz GJ. Induction of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed • 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. • 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. • 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. Table 1. Sub classification of SMA type Subtyp e Age at onset Clinical Features Progression I 0-6 months never sit death by age 2 II 7-18 months sit, but never stand 2 nd -3 rd decade III <30 years may ambulate independently normal lifespan Clinical relevance of Antisense strategy Molecular Genetics of SMN • SMN2 is nearly identical to SMN1 but predominantly aberrantly spliced resulting in decreased amounts of functional SMN. 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). Injection site 4 th ventricle Lateral ventricles O lfactory ventricle A C B D Left Weights of Normal(▲), SMA (■), SMA AO (○), and SMA PEG-PEI-AO (■) from P1-P12. OBJECTIVES • Deliver AO to brain and spinal cord tissue of SMA mice and quantify SMN expression by Western blot. • 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. RESULTS RESULTS RESULTS Test of motor function shows improvement in AO treated SMA mice Right Best righting times of SMA and SMA AO treated mice when placed on either side. Left Chart of best righting times from 3 trials per side for SMA and SMA AO treated mice. Full-length SMN2 measured by real-time PCR •There was a 1.4-fold increase in full-length SMN2 transcripts in SMA AO-treated compared to SMA control. Overview of real-time PCR • TaqMan primers and probes for detecting full-length SMN2 were previously 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. • 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. METHODS 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 4 th ventricle. Dorsal (A, B) and Ventral (C, D) views are shown. 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). Right Increase in SMA AO treated body weight relative to SMA controls on days 9 (*, P<0.01) and 12 (**P<0.05) and SMA PEG-PEI-AO treated showed a significant on day 9 (**P<0.05 ) but not day 12. N=5 for AO and SMA control, N=7 for PEG-PEI-AO treated. Intracerebroventricular injection of fluorescent Antisense Oligonucleotides Quantification of SMN expression by Western Blot SMA Left (sec) Right (sec) Best (sec) Yes/No (1/0) 1 30 30 30 0 2 30 30 30 0 3 30 30 30 0 4 30 30 30 0 5 5 30 5 1 6 4 8 4 1 7 30 30 30 0 8 2 30 2 1 9 30 30 30 0 SMA AO 1 4 5 4 1 2 2 30 2 1 3 3 3 3 1 4 4 30 4 1 5 30 1 1 1 S MN1 S MN2 6 7 8 6 7 8 678 68 100% ~90% ~10% (A) n (A) n (A) n (A) n • 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. SM N expression (% ) H ippocam pus C ervicalSC Lum barSC 0 10 20 30 40 50 60 70 SM A control SM A AO * * ** 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. (100) (18) (41) (41) (28) (27) (26) Tubulin SM N FL Δ7 H ippocam pus A (100) (15) (51) (61) (79) (51) (41) Δ7 FL Tubulin SM N Cervical B (15) (100) (33) (28) (43) (29) (25) FL Δ7 Tubulin SM N Lum bar C wt SM A SM A injected wt SM A SM A injected SM A wt SM A injected Left SMN expression is significantly increased in hippocampus and spinal cord 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). (25) (100) (22) (19) (27) (22) (31) (25) (100) (25) (17) (20) (21) (36) (26) (11) (100) (15) (22) (19) (27) (22) (31) (25) Tubulin SM N Δ7 FL Δ7 FL FL Δ7 Tubulin SM N Tubulin SM N Hippocam pus A wt SM A SM A injected Lum bar C wt SM A SM A injected Cervical B SM A wt SM A injected SM N expression (% ) H ippocam pus C ervicalSC Lum barSC SM N expression (% ) H ippocam pus C ervicalSC Lum barSC 0 5 10 15 20 25 30 SM A control SM A PEG -PEI-AO * 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 PEG-PEI- AO, N=5 for SMA control). Left SMN expression shown for hippocampus (A) brain, cervical (B) and lumbar (C) spinal cord extracts (N=7). FL-SMN and Δ7-SMN are indicated by arrows. Improved SMN expression in Brain and Spinal Cord: AO-saline is more effective than Nanopolymer-AO • 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). Bodyweight analysis of SMA mice from birth-P12 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 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 0 2 4 6 8 10 12 1 2 3 4 5 6 7 8 9 10 11 12 PostnatalD ay W eight (g) non-SM A AO alone PEG -PEI-AO SMA
description
Antisense Mediated Induction of SMN Expression in Brain and Spinal Cord Tissue Improves Bodyweight and Righting Response in the SMA Mouse Jason H Williams 1 , Rebecca C Schray, Xiangying Guan, Carlyn A Patterson, Melanie K Tallent, Gordon J Lutz. - PowerPoint PPT Presentation
Transcript of BACKGROUND
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BACKGROUND
CONCLUSIONS
REFERENCES
Antisense Mediated Induction of SMN Expression in Brain and Spinal Cord Tissue Improves Bodyweight and Righting Response in the SMA Mouse
Jason H Williams1, 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
Le TT, Pham LT, Butchbach ME, Zhang HL, Monani UR, Coovert DD, Gavrilina TO, Xing L, Bassell GJ, Burghes AH (2005) SMNDelta7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN. Hum.Mol.Genet. 14: 845-857
Singh NK, Singh NN, Androphy EJ, Singh RN (2006) Splicing of a critical exon of human Survival Motor Neuron is regulated by a unique silencer element located in the last intron. Mol.Cell Biol. 26: 1333-1346
Sumner CJ, Kolb SJ, Harmison GG, Jeffries NO, Schadt K, Finkel RS, Dreyfuss G, Fischbeck KH (2006) SMN mRNA and protein levels in peripheral blood: biomarkers for SMA clinical trials. Neurology 66: 1067-73
Williams JH, Schray RC, Sirsi SR, Lutz GJ. Nanopolymers improve delivery of exon skipping oligonucleotides and concomitant dystrophin expression in skeletal muscle of mdx mice. BMC Biotechnology 2008 Apr 2;8(1):35.
Williams JH, Sirsi SR, Latta DR, Lutz GJ. Induction of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed with PEG-PEI copolymers. Molecular Therapy 2006 Jul; 14(1):88-96
Wirth B, Brichta L, Schrank B, Lochmuller H, Blick S, Baasner A, Heller R (2006) Mildly affected patients with spinal muscular atrophy are partially protected by an increased SMN2 copy number. Hum.Genet. 119: 422-428
• 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.
• 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.
• 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.
Table 1. Sub classification of SMA type
Subtype Age at onset Clinical Features ProgressionI 0-6 months never sit death by age 2
II 7-18 months sit, but never stand 2nd-3rd decade
III <30 years may ambulate independently normal lifespan
Clinical relevance of Antisense strategy
Molecular Genetics of SMN• SMN2 is nearly identical to SMN1 but predominantly aberrantly
spliced resulting in decreased amounts of functional SMN.
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).
Injectionsite
4th ventricle
Lateral ventricles
Olfactoryventricle
A
C
B
D
Left Weights of Normal(▲), SMA (■), SMA AO (○), and SMA PEG-PEI-AO (■) from P1-P12.
OBJECTIVES
• Deliver AO to brain and spinal cord tissue of SMA mice and quantify SMN expression by Western blot.
• 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.
RESULTS
RESULTS RESULTS
Test of motor function shows improvement in AO treated SMA mice
Right Best righting times of SMA and SMA AO treated mice when placed on either side.
Left Chart of best righting times from 3 trials per side for SMA and SMA AO treated mice.
Full-length SMN2 measured by real-time PCR• There was a 1.4-fold increase in full-length SMN2 transcripts in
SMA AO-treated compared to SMA control.
Overview of real-time PCR• TaqMan primers and probes for detecting full-length SMN2 were previously
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.
• 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.
METHODS
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.
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).
Right Increase in SMA AO treated body weight relative to SMA controls on days 9 (*, P<0.01) and 12 (**P<0.05) and SMA PEG-PEI-AO treated showed a significant on day 9 (**P<0.05 ) but not day 12. N=5 for AO and SMA control, N=7 for PEG-PEI-AO treated.
Intracerebroventricular injection of fluorescent Antisense Oligonucleotides
Quantification of SMN expression by Western Blot
SMA Left(sec)
Right(sec)
Best(sec)
Yes/No (1/0)
1 30 30 30 0
2 30 30 30 0
3 30 30 30 0
4 30 30 30 0
5 5 30 5 1
6 4 8 4 1
7 30 30 30 0
8 2 30 2 1
9 30 30 30 0SMA
AO
1 4 5 4 1
2 2 30 2 1
3 3 3 3 1
4 4 30 4 1
5 30 1 1 1
SMN1
SMN2
6 7 8
6 7 8
6 7 8
6 8
100%
~90%
~10%
(A)n
(A)n (A)n
(A)n
• 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.
SM
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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.
(100) (18) (41) (41) (28) (27) (26)
Tubulin
SMN FLΔ7
HippocampusA
(100) (15) (51) (61) (79) (51) (41)
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(15) (100) (33) (28) (43) (29) (25)
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wt SMA SMA injected
wt SMASMA injected
SMA wtSMA injected
Left SMN expression is significantly increased in hippocampus and spinal cord 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).
(25) (100) (22) (19) (27) (22) (31) (25)
(100) (25) (17) (20) (21) (36) (26) (11)
(100) (15) (22) (19) (27) (22) (31) (25)
Tubulin
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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 PEG-PEI-AO, N=5 for SMA control).
Left SMN expression shown for hippocampus (A) brain, cervical (B) and lumbar (C) spinal cord extracts (N=7). FL-SMN and Δ7-SMN are indicated by arrows.
Improved SMN expression in Brain and Spinal Cord: AO-saline is more effective than Nanopolymer-AO
• 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).
Bodyweight analysis of SMA mice from birth-P12
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
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).
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