Development of histone deacetylase inhibitors Development of histone deacetylase inhibitors as therapeutics for Friedreich’s ataxiaas therapeutics for Friedreich’s ataxia

http://www.scripps.edu/mb/gottesfeld

Joel Gottesfeld

Department of Molecular BiologyThe Scripps Research InstituteLa Jolla, California USA

National Ataxia FoundationSymposiumLos Angeles, CAMarch 19, 2011

Silencing of the frataxin FXN gene in Friedreich’s ataxia patient cells is due to expanded GAA.TTC repeats:

Taken from: http://biol.lf1.cuni.cz/ucebnice/en/non-mendelian_heredity.htm

Since the GAA repeats do not change the coding potential of the frataxin gene, gene activation would be a therapeutic strategy.But, how do the repeats cause the FXN gene to be silenced?

Friedreich’s ataxia

Unaffected

Triplet repeat DNA can form unusual DNA structures, which mayinterfere with gene transcription:

Biochemical experiments with GAA repeat DNA show that it can form atriple-stranded structure, which blocks RNA polymerase from copying the sequence of DNA into RNA, leading to a loss of frataxin protein

However, no experiments have been done to prove that this structure actuallyexists in cells from FRDA patients….so, this is just a hypothesis.

Double-stranded DNA Triplex DNA

Nucleosome

DNA

30 nm fiber

‘Higher orderstructure’

‘Nuclear domains’

Chromatin organization is modularAlternatively, the repeats can change FXN

chromatin structure to silence the gene: levels of chromatin organization in the cell nucleus

Loss of frataxin mRNA in FRDA may be due to a repressive chromatin structure -- Whether a gene is active or not depends on small chemical tags that are attached to the histone proteins….acetylation and methylation

Silencing of the frataxin FXN gene by expanded GAA.TTC repeats in FRDA is due to condensation of the gene into a form of chromatin, called heterochromatin, that blocks copying the gene into messenger RNA. This results in a loss of frataxin protein compared to healthy individuals.

- Heterochromatin is controlled by a class of enzymes call histone deacetylases or HDACs that remove the acetyl groups from the histones

- HDAC inhibitors reverse FXN silencing by directly increasing acetylation, which leads to chromatin decondensation and more frataxin protein

Taken from: Festenstein, Nature Chem. Biol. 2006

4c/BML-210 4b

Western blot: frataxin protein FXN mRNA +/- 4b

OO

HN NHNH2 OOHN NHNH2

Ryan Burnett/Sue Perlman, UCLA

Rel

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5 M

5.0 M

7.5

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ated

2.5 M

5.0 M

7.5

MU

ntre

ated

2.5 M

5.0 M

7.5

M

carrier D patient J patient M

Reversal of the transcription defect to at leastReversal of the transcription defect to at leastcarrier status in PBMCs from >50 individualscarrier status in PBMCs from >50 individuals

HDAC inhibitors tested for effects on FXN expression in white blood cells(primary lymphocytes) from FRDA patients:

Myriam Rai/Massimo Pandolfo/Giovanni Coppola/Dan Geschwind

OO

HN

HN

NH2

106

Mouse model for Friedreich’s ataxia (Pandolfo laboratory, Brussels): KIKI mice

- Knock-in of GAA repeats in intron 1 of the mouse frataxin gene results in decreased mRNA

- HDACi 106 crosses the blood-brain barrier and increases histone acetylation in the brain

- HDACi 106 restores brain and heart FXN mRNA levels to that of normal mice

100

151143

174159

177

0

50

100

150

200

250

KIKI WT

vehicle

4b

106

Relative fxn mRNA expression

Acetylated Histone 3

Total Histone 3

Vehicle 106

• Compound 106, when injectedsubcutaneously, crosses theblood-brain barrier and inhibitsHDACs in vivo in the KIKI brain

KIKI WT

Induced pluripotent stem cells offer promise for generating neuronal models

for FRDA

subject

Taken from Sigma/Aldrich

mixed population of retroviruses

GM03816 FA patient fibroblasts

~30 days

O/N

Takahashi et al, Cell 131, 861.

H1 human ES cells

KLF4

Sox2 c-Myc

Oct4

Development of a human neuronal cell model for FRDA: Reprogramming of patient cells via retroviral delivery to generate induced pluripotent stem (iPS) cells

Sherman Ku

FRDA iPS cellsSelection based oncolony morphology

i iii iv

v vi vii

viii ix x

i ii iii iv

v vi vii

viii ix x

Reprogramming of patient cells–characterization by immunofluorescence, teratoma formation, global and FXN gene expression, FXN chromatin, GAA repeats, etc.

Illumina gene expression profiling of Friedreich’s ataxiaiPS cells versus other iPS and ES cells, as well astissues and cell lines (Loring lab, TSRI): evidence forpluripotency plus a disease signature

Ku et al., Cell Stem Cell, 2010

Directed in vitro neuronal differentiation:

Dottori and Pera, Methods Mol. Biol. 438, 19, 2008.

H1 or iPScells

noggin induction (14 days)large colonies form (nestin positive), still retain normal colony morphology

formation of neurospheres, contains neural stem cells or precursors; rosettes can be visible.

dissect and passage as suspension culture

replate to adherent culture and allow

for maturation

Immunostaining for beta-III tubulin (Tuj1), an early neuronal marker~85 – 95% pure cells byFACS analysis

FRDA neuronal cells retain FXN gene silencing: FXN mRNA and frataxin protein levels comparable in neuronal cellsas in the parent FRDA iPS cells.

FXN mRNA Frataxin protein

FRDA neuronal cells also show heterochromatin marks as inhuman cells. Will these cells respond to HDAC inhibitors?

Sherman Ku

HDAC3-selective inhibitor 109 increases FXN mRNA and frataxin protein levels:

Sherman Ku

Testing both HDAC3 and HDAC1/2 selective compound for increases in FXN mRNA levels (109: ~5-fold HDAC3 selective; 3: 300-fold HDAC1/2 selective)

RG109 increases frataxin levels by ~2-fold, a therapeutically useful increase!

FXN mRNA Frataxin protein

FRDA neuronal cells have a defect in mitochondrial activity, asdetermined with a dye that measures mitochrondrial membrane potential. Compare unaffected and FRDA neurons.

HDACi 109 restores mitochondrial activity to unaffected levels.

Sherman Ku/Erica Campau

Average of five determinations, with standard deviations;Values normalized to unaffected control neurons

Will our HDAC inhibitors lead to human therapeutics?

Repligen Corporation of Waltham, MA, has licensed our HDAC inhibitors for development:

-- compounds tested for drug-like properties, found to be ok

-- a large library of derivatives were synthesized and active molecules identified

-- improved compounds identified – more active and less toxic to cells

-- Pre-clinical development (full pharmacology and toxicology) completed for a clinical candidate (RG2833)

-- IND filed with the FDA and phase I safety trials in human subjects to commence soon

What do we expect to learn from a phase I safety trial?

-- whether the compounds are safe and well-tolerated in humans

-- whether they increase the levels of frataxin in white blood cells from treated patients

-- This latter result would be a “proof-of-principal” showing that full phase II studies should be done (very costly!)

Summary:

- Friedreich’s ataxia is a chromatin disease: GAA repeats induce heterochromatin mediated gene silencing, through histone modifications

- Novel HDAC inhibitors relieve FXN gene silencing by directly increasing histone acetylation on pathogenic FXN alleles, both in FRDA patient cells and in a mouse model

- FRDA iPSCs mimic FXN gene silencing

- FRDA iPSCs can be differentiated into neurons in vitro, providing a new cellular model for FRDA

- FRDA neuronal cells respond to our HDAC inhibitors, suggesting that they may be beneficial in the human disease

Ryan Burnett Ryan Burnett Chunping Xu Chunping Xu James Chou James Chou Fang Hu Fang HuDavid HermanDavid Herman Christine Christine JespersenJespersenKai JenssenKai Jenssen Erica Campau Erica CampauSherman KuSherman Ku Louise Laurent Louise LaurentElisabetta Soragni Jeanne LoringElisabetta Soragni Jeanne LoringJintang DuJintang Du

Scripps:Scripps: UCLA:UCLA: Susan PerlmanSusan Perlman Giovanni CoppolaGiovanni Coppola Dan GeschwindDan Geschwind

Brussels:Brussels: Myriam RaiMyriam Rai Massimo PandolfoMassimo Pandolfo

Repligen:Repligen: Jim Rusche and colleaguesJim Rusche and colleagues

London:London: Mark Pook and colleaguesMark Pook and colleagues

www.scripps.edu/mb/gottesfeld

With support from:With support from:

Friedreich’s Ataxia Research AllianceFriedreich’s Ataxia Research Alliance

National Institute of NeurologicalNational Institute of NeurologicalDisorders and Stroke (NINDS/NIH)Disorders and Stroke (NINDS/NIH)

GoFAR/Ataxia UK/Ataxia IrelandGoFAR/Ataxia UK/Ataxia Ireland

Repligen CorporationRepligen Corporation