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Valproic acid in SMA
Brunhilde WirthInstitute of Human Genetics
University of Cologne, Germany
DNA Exon 1 Intron 1 Exon 2 Exon 3Intron 2
AGTTTTAGA GACTTCAGA ACTAGACAGT...AG GT…AG
AGUUUUAGA GACUUCAGA ACUAGACAGU…AG GU…AG
Prä-mRNA Transkription
S F R D F R T R Q
Protein Translation
AGUUUUAGA ...GACUUCAGA ACUAGACAA...
mRNASpleißen
From Gene to protein
mRNA Protein
SMN genes, transcripts and proteins
1 -5 6 7 8
1 -5 6 8
FL-SMN
SMN7
100% SMN1 10% SMN2
90% SMN2
DNA pre-mRNA
Exons 1 2a 2b 3 4 5 6 7 8
E6 E8
Alternative splicing of SMN2 RNA
…ttattttccttacagGGTTTCAGACAAAATCAAAAAGAAGGAAGG…
SMN1
SMN2
…AGgt
Splice donor Splice aceptor
GGTTTTAGACAAAATCAAAAAGAAGGAAGG…
Exonic splicing enhancer
SF2-ASF
SF2-ASF
Cartegni & Krainer, Nat Genet 2002
Correct splicing of SMN2 is restored by overexpression of Htra2-β1 and Hra2-β1
interacting splicing factors
Hofmann et al., PNAS 2000; Hofmann and Wirth; Hum Mol Genet 2002
Htra2-β1
pSMN2 Minigen
6 7 8
*
SE1 SE2 SE3
GGUUUCCAGACAAAAUCAAAAAGAAGGAAGGUGCUCACAUUCCUUAAAUUAAGGA
pSMN2
Htra2-β1
0 1 3 5
4x
Increase of
FL-SMN2
FL-SMN
Δ7-SMNhnRNP-G
SRp30c
How to increase the SMN2 protein expression ?
SMN2FL-SMN20%
SMN780%
#1: transcriptional activation of SMN2
SMNSMN
SMN
SMN
2x increase
Therapeutical strategies for SMA
SMN2
#2: correction of SMN2 splicing pattern
FL-SMN20%
SMN780%
SMNSMN
SMN
SMNFL-SMN40%
SMN740%
Htra2-ß1
COOH
NN
H
H
OH
O
O
HDAC inhibitors
1. Short chain fatty acids
-Valproic acid (VPA)
-Phenylbutyrate (BP)
-Sodium butyrate (NaBu)
2. Hydroxamic acids
-TSA
-SAHA
3. Benzamides
-MS275
-M344
SAHA
VPA
M344
HDAC inhibitors activate gene transcription
transcription
no methylationacetylation
methylationno acetylation
HDAC inhibitors
Rational for valproic acid as a potential therapy for SMA
• Treatment of SMA with butyrate Chang et al. PNAS (2001)
short chain fatty acid, HDAC inhibitorBUT: very short half life in vivo (6 min in serum)
• Valproic acid (VPA): another short chain fatty acid, HDAC inhibitor Phiel et al. J Biol Chem (2001), Göttlicher et al. Embo J (2001)
FDA approved drug, successfully used in epilepsy treatment for decades, half life 8 to 10 h in human serum
COOH
SMNmaximum
VPA increases SMN protein levels depending on the SMN2 copy number
β-Tubulin
SMN
fibroblast line ML-17
SMA I2 SMN2 copies
Moc
k
0.5
µM
5 µM
50 µ
M
500
µM
1000
µM
fibroblast line ML-16
SMA I3 SMN2 copies
Moc
k
0.5
µM
5 µM
50 µ
M
500
µM
1000
µM
fibroblast line ML-5
SMA II3 SMN2 copies
Moc
k
0.5
µM
5 µM
50 µ
M
500
µM
1000
µM
2.7x 3.1x 4.3x1.8x 3.0x 3.6x
Brichta et al., Hum. Mol. Genet. 2003,19, 2481-2489
SMNmaximum
VPA mediates reversion of Δ7-SMN2 RNA into FL-SMN2 RNA
via Htra2-β1
Htra2-β1maximum 2.7x 2.7x
β-Tubulin
Htra2- β1
Moc
k
0.5
µM
5 µM
50 µ
M
500
µM
1000
µM
fibroblast line ML-17
Moc
k
0.5
µM
5 µM
50 µ
M
500
µM
1000
µM
fibroblast line ML-16
Moc
k
0.5
µM
5 µM
50 µ
M
500
µM
1000
µM
fibroblast line ML-5
SMA I2 SMN2 copies
SMA I3 SMN2 copies
SMA II3 SMN2 copies
Brichta et al., Hum. Mol. Genet. 2003,19, 2481-2489
4.1x
• The SMN2 transcription level is increased by promotor activation most likely through AP1 and Sp1 elements
• Δ7-SMN2 RNA is restored into FL-SMN2 RNA most likely due to increased levels of Htra2-ß1
Mechanism of action of valproic acid on the SMN2 gene
Brichta et al., Hum. Mol. Genet. 2003,19, 2481-2489
ß-actin (42 kDa)
rat smn (40 kDa)
ß-actin (42 kDa)
human SMN (40 kDa)
controlVPA
RAT hippocampal brain slices HUMAN hippocampal brain slicesderived from epilepsy surgery
controlVPA
Drug validation
Brichta et al., Hum Mol Genet. 2003; Hahnen et al., submitted
SAHA increases SMN protein level in SMA fibroblasts
β-Tubulin
SMN
3.0xSMN
maximum
SAHA
NN
H
H
OH
O
O
fibroblast line ML-16
SMA I3 SMN2 copies
Moc
k
0.05
µM
0.5
µM
1 µM
5 µM
10 µ
M
24h
Hahnen et al., submitted
- Transcription activation
- Very modest correction of splicing
Human brain slices derived from epilepsy surgery
-actin (42 kDa)
human SMN (40 kDa)
contro
l
16 µ
M
32 µ
M
64 µ
M
128
µM2
mM
VPA
SAHA
P1
P2
Drug validation
Treatment of SMA fibroblasts with M344
M344
Hahnen et al., submitted
β-Tubulin
SMN
3xSMN
maximum
fibroblast line ML-16
SMA I3 SMN2 copies
64h
Moc
k
0.5
µM 5
µM
10 µ
M
30 µ
M
50 µ
M
100
µM
Treatment of rat cultured motor neurons with
VPA, M344 and SAHA
Hahnen et al., submitted
Clinical trial with VPA in SMA parents
• 10 parents of type I-III SMA patients • All have one SMN1 and 1-3 SMN2 copies. • Treatment for about 4-5 months (70-80 µg VPA/ml
serum)• 9 measurements of SMN2-RNA and SMN-protein
from blood samples
Results:
6/10 showed an increase of FL-SMN between 40-300%
• Therapy with VPA in SMA patients
• Individual therapies of about 40 type I-III SMA patients (seen by various neuropediatricians, neurologists, pediatricians in Germany and Austria)
• Patients have a level of VPA in serum of ~70-80µg/ml• Most patients were also substituted with L-carnitin (50-100
mg/kg/day) • The younger the patients are the more improvements were
reported• First improvements usually after ~6 months of therapy• There were no significant negative side effects reported so
far • Results of quantitative measurements of FL-SMN showed an
up-regulation in about 50% of patients
Individual therapy of SMA patients with VPA
Individual therapy of SMA patients with VPA
Future trials with VPA
• Germany: type I SMA patients – Extinction on European level
• USA: type II and III SMA patients – Starts in June 2005
• EU: type II SMA patients– Planed, next meeting of the ENMC in autumn
CologneLars BrichtaEric Hahnen Markus Riessland Irmgard Hölker
BonnYvonne Hofmann Karsten HaugYuli SunHeidrun Raschke Thomas KlockgetherSebastian Stier
Initiative“Forschung und Therapie
für SMA”SMA-Germany
ErlangenFlorian SiebzehnrublIngmar BlümckeIlker Eyupoglu
HannoverKirsten HaastertPeter Claus
Köln Fortune
Köln Fortune
ZMMK