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Targeting the GAA-Repeat Region with Oligonucleotides for the Treatment of Friedreich’s Ataxia Fatih Ozsolak1, Kamaljeet Sandhu1, Susan Wood1,Mark Wysk1, Paula Lewis1, David Bullough1,James Barsoum1 1RaNA Targeting the repeat region in both directions with oligonucleotides lead to FXN upregulation in diseased fibroblasts Abstract Friedreich’s ataxia (FRDA) is a recessively inherited neuromuscular disorder that arises due to cellular depletion of frataxin (FXN) protein and resulting defects in mitochondrial functions. The protein coding sequence of FXN is normal in the majority of FRDA patients, suggesting that upregulation of endogenous FXN expression could be an effective therapy. The most common molecular cause of this disease is the expansion of GAA/TTC triplet repeats in the first intron of FXN gene. Repeat expansion beyond a certain threshold causes transcriptional defects which reduce FXN mRNA and protein levels. Despite long-standing research in the pathogenesis of FRDA, the means by which GAA-repeat number elevation leads to transcriptional silencing is not clear. DNA-DNA and DNA-RNA interactions formed in the long triplet repeat stretches, defects and alterations in splicing patterns and the formation of a heterochromatin-like structure are among the hypotheses being considered. In order to gain clues into the mechanisms responsible for the FXN deficit in FRDA, we undertook genome-wide analyses to examine the global and local RNA species and chromatin structure and composition changes in FRDA patient cells. Epigenetic screens identified two chromatin modifying complexes as being important in establishing and/or maintaining repeat expansion-induced transcriptional repression at the FXN locus. We identified a novel putative non-coding RNA (ncRNA) potentially responsible for directing the localized epigenetic silencing of the FXN gene. To target this novel ncRNA, we have used locked nucleic acid “gapmer” oligonucleotides consisting of LNA ends and a central DNA stretch to degrade the putative ncRNA by an RNase H-mediated process. Targeting this ncRNA led to FXN mRNA and protein upregulation at therapeutically significant levels in FRDA patient cells in vitro and a FRDA mouse model. The oligonucleotide-based therapeutic approaches developed here pave the way towards the design of multiple strategies for the treatment of FRDA and may have applications for the treatment of other human diseases. Friedreich’s Ataxia (FRDA) mRNA targets HO O • Oligonucleotides targeting the repeat region may act to reverse heterochromatin formation by interfering with a currently uncharacterized heterochromatin formation mechanism in humans. • In vivo studies using humanized FRDA mouse models are ongoing to assess FXN mRNA/protein level changes in response to oligo treatment. The CNS is being targeted by intrathecal delivery. Heart is being targeted by subcutaneous dosing of oligos. • Future mechanistic studies will focus on characterizing potential RNAs present at/near the repeat region and understanding oligo mechanism of action through epigenetic and posttranscriptional alterations. to C. Figure 2. (A) ChIP analyses in regions surrounding the expanded repeat suggest heterochromatic marks to be present in repeat neighboring “unique” regions, in line with previous findings (De Biase et al. PLoS ONE. 2009). FXN mRNA upregulation with gapmers (B,C) targeting “unique” regions and with a GAA-targeting mixmer (D) in three diseased fibroblast lines via transfection. GAA repeat numbers: GM03665 (816,1410), GM03816 (641, 480) and UAB68 (570,1200). UAB68 cell line was kindly provided by Dr. Marek Napierala. (E) Mature FXN protein upregulation with RaNA-7188 and -7189 in GM03816 cells.. D. RNA End Targeting Platform to Modulate Gene Expression (A) RNA degradation can take place via exonucleases that chew RNAs from ends, or via endonucleases. Targeting RNA ends with oligos may decrease RNA degradation, increase stability and RNA/protein levels. Targeting of RNA ends with oligos may lead to gene upregulation via other mechanisms too. (B) Pseudo-circularization of RNA via oligos may achieve stability and/or translation efficiency increase. Oligos can be designed with various extensions to protect polyA tail and 5’ cap (C,D). B. mRNA A. Potential RNA presence in the repeat and neighboring regions that may be targeted by the AGO2 machinery LNA mixmer oligo B. 5’ end 3’ end A AAA polyA tail Gatchel & Zoghbi. Nature Reviews Genetics 2005 C. Mixmers (15mers and shorter) O Multiple oligonucleotides have been discovered upregulate FXN mRNA and protein levels in vitro. A. B ASO Gapmers (13-16mers) • B. E. Oligotherapeutics Technologies HO A. Conclusions AAAAA A A • The most common inherited ataxia (1:50,000 WW); autosomal recessive • Progressive degenerative neuromuscular disorder with onset varies after 5yrs • Frataxin, the gene implicated in FRDA, is highly expressed in heart, brain, spinal cord and voluntary skeletal muscle • Frataxin likely function in the formation of ironsulphur cluster-containing proteins and iron methabolism • GAA repeat expansion in Frataxin intron 1 results in reduced mRNA production via heterochromatin-like structure formation and/or triplex DNA structure formation • In most cases, frataxin exons are not mutated. Therefore, increased expression of the endogenous gene should be curative Therapeutics, Inc., Cambridge, MA miRNA, lncRNA targets Compounds RaNA aims to develop as therapeutic agents are short singlestranded, stabilized oligos. Oligos can be used as “gapmers” that act to degrade target RNAs via recruitment of RNAse-H mechanism, or as “mixmers” that function as steric blockers. The different activities of mixmer and gapmer oligos are due to different positioning of modified nucleotides. RaNA primarily uses the Locked Nucleic Acid (LNA) chemistry due to their increased potency and improved drug-like properties involving : Figure 3. (A) Rnase protection assay (RPA) C. analyses using a short 24-mer GAA probe using small RNA fraction from the indicated diseased and normal cell lines suggest a protected RNA being present that may be inversely correlated with FXN mRNA levels in lymphoblast cell lines. (B) Early RPA analyses using a long RNA probe (shown in panel C) D. targeting the antisense strand suggest protected RNA species to be present. This probe overlaps with the previously described FAST-1 transcript (De Biase et al. PLoS ONE. 2009). Current studies are exploring the boundaries and character of these protected species. (D) AGO2 gapmer upregulates FXN mRNA in GM03816 cells. Improved nuclease resistance; long tissue half-life Preliminary evidence for FXN mRNA and protein upregulation with gapmers targeting the repeat region in vitro and in vivo More potent; have higher affinity for mRNA (increases melting temp by 46oC per base) Reduced immune stimulation No delivery system needed – administration in saline by subcutaneous injection leads to broad tissue biodistribution A. B. D. RNA End Targeting Platform to Modulate Gene Expression Dose-dependent FXN protein upregulation with 5’ and 3’ oligos (A) and pseudocircularization oligos (B) in GM3816 cells via transfection. FXN mRNA upregulation in GM3816 cells with 5’ and 3’ oligos (C), and in the liver of Sarsero mouse model after oligo treatment via subcutaneous injection (D). A. A. Epigenetic siRNA screen identifies NuA4 Histone Acetyl-transferase Complex as potential regulator of FXN repression B. B. Figure 1. (A) An RNAi screen with 464 siRNAs against major epigenetic genes were performed. The results confirmed FXN downregulation in response to CTCF knockdown (>5-fold, De Biase et al. PLoS ONE. 2009). The FXN fold changes obtained at different times in two diseased cell lines are very similar. (B) Gene pathway analysis of genes affecting FXN levels in response to siRNA treatment shows an enrichment for NuA4 histone acetyltransferase complex genes. Figure 4. Gapmer RaNA 4376 was C. transfected to GM03816 cells. Transfections were repeated on day4 and day8, and cells were split as they became confluent. FXN mRNA levels (A) were measured with quantitative RT-PCR in the indicated timepoints. FXN protein levels (B) were measured using a commercial ELISA kit. (C) Sarsero FRDA mice were dosed subcutaneously with RaNA 4376 (n=6) for 8 weeks with dosing every other week. The RNA data was normalized to the control group and using three housekeeper genes. A statistically significant increase in FXN was seen in heart. C. D.