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

1. 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

2. 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

3. 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

4. Nucleosome 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 orderstructure’ ‘Nuclear domains’ 30 nm fiber DNA

5. 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

6. 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

7. patient M patient J carrier D Relative Frataxin mRNA 5.0 mM 2.5 mM 7.5mM 2.5 mM 2.5 mM Untreated Untreated Untreated 7.5mM 5.0 mM 7.5mM 5.0 mM HDAC inhibitors tested for effects on FXN expression in white blood cells (primary lymphocytes) from FRDA patients: Western blot: frataxin protein FXN mRNA +/- 4b 4c/BML-210 4b Reversal of the transcription defect to at least carrier status in PBMCs from >50 individuals Ryan Burnett/Sue Perlman, UCLA

8. 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 KIKI WT Myriam Rai/Massimo Pandolfo/Giovanni Coppola/Dan Geschwind

9. Induced pluripotent stem cells offer promise for generating neuronal models for FRDA subject Taken from Sigma/Aldrich

10. Development of a human neuronal cell model for FRDA: Reprogramming of patient cells via retroviral delivery to generate induced pluripotent stem (iPS) cells KLF4 c-Myc Sox2 Oct4 O/N mixed population of retroviruses 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.

11. Reprogramming of patient cells–characterization by immunofluorescence, teratoma formation, global and FXN gene expression, FXN chromatin, GAA repeats, etc. i iii iv v vi vii viii ix x i ii iii iv v vi vii Illumina gene expression profiling of Friedreich’s ataxia iPS cells versus other iPS and ES cells, as well as tissues and cell lines (Loring lab, TSRI): evidence for pluripotency plus a disease signature viii ix x Ku et al., Cell Stem Cell, 2010

12. Directed in vitro neuronal differentiation: large colonies form (nestin positive), still retain normal colony morphology noggin induction (14 days) H1 or iPS cells dissect and passage as suspension culture replate to adherent culture and allow for maturation formation of neurospheres, contains neural stem cells or precursors; rosettes can be visible. Immunostaining for beta-III tubulin (Tuj1), an early neuronal marker ~85 – 95% pure cells by FACS analysis Dottori and Pera, Methods Mol. Biol.438, 19, 2008.

13. FRDA neuronal cells retain FXN gene silencing: FXN mRNA and frataxin protein levels comparable in neuronal cells as in the parent FRDA iPS cells. Frataxin protein FXN mRNA FRDA neuronal cells also show heterochromatin marks as in human cells. Will these cells respond to HDAC inhibitors? Sherman Ku

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

15. 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

16. 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!)

17. 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

18. UCLA: Susan Perlman Giovanni Coppola Dan Geschwind Brussels: Myriam Rai Massimo Pandolfo Repligen: Jim Rusche and colleagues London: Mark Pook and colleagues Scripps: Ryan Burnett Chunping Xu James Chou Fang Hu David Herman Christine Jespersen Kai Jenssen Erica Campau Sherman Ku Louise Laurent Elisabetta Soragni Jeanne Loring Jintang Du With support from: Friedreich’s Ataxia Research Alliance National Institute of Neurological Disorders and Stroke (NINDS/NIH) GoFAR/Ataxia UK/Ataxia Ireland Repligen Corporation www.scripps.edu/mb/gottesfeld