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