Development of histone deacetylase inhibitors
as therapeutics for Friedreich’s ataxia
Joel Gottesfeld
Department of Molecular Biology
The Scripps Research Institute
La Jolla, California USA
National Ataxia Foundation
Symposium
Los Angeles, CA
March 19, 2011
http://www.scripps.edu/mb/gottesfeld
Silencing of the frataxin FXN gene in Friedreich’s ataxia patient cells
is due to expanded GAA.TTC repeats:
Unaffected
Friedreich’s ataxia
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?
Taken from: http://biol.lf1.cuni.cz/ucebnice/en/non-mendelian_heredity.htm
Triplet repeat DNA can form unusual DNA structures, which may
interfere with gene transcription:
Biochemical experiments with GAA repeat DNA show that it can form a
triple-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 actually
exists in cells from FRDA patients….so, this is just a hypothesis.
Double-stranded DNA
Triplex DNA
Alternatively, the repeats can change FXN chromatin
structure to silence the gene: levels of chromatin
organization in the cell nucleus
Chromatin organization is modular
‘Higher order
structure’
‘Nuclear
domains’
30 nm fiber
Nucleosome
DNA
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.
HDAC inhibitors tested for effects on FXN expression in white blood cells
(primary lymphocytes) from FRDA patients:
Western blot: frataxin protein
FXN mRNA +/- 4b
patient J
patient M
Relative Frataxin mRNA
carrier D
NH2
O
HN
HN
NH
O
4c/BML-210
NH2
NH
O
O
4b
Ryan Burnett/Sue Perlman, UCLA
Reversal of the transcription defect to at least
carrier status in PBMCs from >50 individuals
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
Relative fxn mRNA expression
Vehicle 106
Acetylated
Histone
250
3
200
174
159
Total
Histone
3
143
150
177
151
vehicle
4b
100
106
100
¥ Compound
106, when injected
subcutaneously, crosses the
blood-brain barrier and inhibits
HDACs in vivo in the KIKI brain
50
0
KIKI
KIKI
H
N
106
H
N
O
WT
WT
NH2
O
Myriam Rai/Massimo Pandolfo/Giovanni Coppola/Dan Geschwind
Induced pluripotent stem cells offer promise for
generating neuronal models for FRDA
subject
Taken from Sigma/Aldrich
Development of a human neuronal cell model for FRDA: Reprogramming of
patient cells via retroviral delivery to generate induced pluripotent stem (iPS) cells
O/N
KLF4
mixed population of
retroviruses
Sox2
c-Myc
Oct4
GM03816 FA patient fibroblasts
H1 human ES cells
~30 days
FRDA iPS cells
Selection based on
colony morphology
Sherman Ku
Takahashi et al, Cell 131, 861.
Reprogramming of patient cells–characterization by
immunofluorescence, teratoma formation, global and FXN
gene expression, FXN chromatin, GAA repeats, etc.
i
iii
v
vi
vii
viii
ix
x
i
ii
iii
v
vi
vii
Illumina gene expression profiling of Friedreich’s ataxia
iPS cells versus other iPS and ES cells,
as well asix
viii
tissues and cell lines (Loring lab, TSRI): evidence for
pluripotency plus a disease signature
Ku et al., Cell Stem Cell, 2010
x
iv
iv
Directed in vitro neuronal differentiation:
H1 or iPS
cells
noggin induction (14 days)
large colonies form
(nestin positive), still
retain normal colony
morphology
dissect and passage
as suspension
culture
replate to adherent
culture and allow
for maturation
Immunostaining for betaIII tubulin (Tuj1), an early
neuronal marker
~85 – 95% pure cells by
FACS analysis
formation of
neurospheres,
contains neural
stem cells or
precursors;
rosettes can be
visible.
Dottori and Pera, Methods Mol. Biol. 438, 19, 2008.
FRDA neuronal cells retain FXN gene silencing:
FXN mRNA and frataxin protein levels comparable in neuronal cells
as in the parent FRDA iPS cells.
FXN mRNA
Frataxin protein
FRDA neuronal cells also show heterochromatin marks as in
human cells. Will these cells respond to HDAC inhibitors?
Sherman Ku
HDAC3-selective inhibitor 109 increases FXN mRNA and
frataxin protein levels:
FXN mRNA
Frataxin protein
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!
Sherman Ku
FRDA neuronal cells have a defect in mitochondrial activity, as
determined with a dye that measures mitochrondrial membrane
potential. Compare unaffected and FRDA neurons.
HDACi 109 restores mitochondrial activity to unaffected levels.
Average of five determinations, with standard deviations;
Values normalized to unaffected control neurons
Sherman Ku/Erica Campau
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
UCLA:
Scripps:
Ryan Burnett
James Chou
David Herman
Kai Jenssen
Sherman Ku
Elisabetta Soragni
Jintang Du
Chunping Xu
Fang Hu
Christine Jespersen
Erica Campau
Louise Laurent
Jeanne Loring
With support from:
Friedreich’s Ataxia Research Alliance
Susan Perlman
Giovanni Coppola
Dan Geschwind
Brussels:
Myriam Rai
Massimo Pandolfo
Repligen:
Jim Rusche and colleagues
London:
Mark Pook and colleagues
National Institute of Neurological
Disorders and Stroke (NINDS/NIH)
GoFAR/Ataxia UK/Ataxia Ireland
Repligen Corporation
www.scripps.edu/mb/gottesfeld