development of histone deacetylase inhibitors as therapeutics for friedreich’s ataxia joel...
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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
ativ
e F
rata
xin
mR
NA
Unt
reat
ed2.
5 M
5.0 M
7.5
MU
ntre
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
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