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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
Targeting the repeat region in both directions with
oligonucleotides lead to FXN upregulation in
diseased fibroblasts
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
Oligonucleotides targeting the repeat region may act to
reverse heterochromatin formation by interfering with a
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.
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
upregulation with RaNA-7188 and -7189 in
GM03816 cells..
RNA End Targeting Platform to Modulate Gene
(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).
Potential RNA presence in the repeat and
neighboring regions that may be targeted by the
AGO2 machinery
LNA mixmer oligo
5’ end
3’ end
polyA tail
Gatchel & Zoghbi. Nature Reviews Genetics 2005
Mixmers (15mers and shorter)
Multiple oligonucleotides have been discovered
upregulate FXN mRNA and protein levels in vitro.
ASO Gapmers (13-16mers)
Oligotherapeutics Technologies
• 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
• 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)
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
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).
Epigenetic siRNA screen identifies NuA4 Histone
Acetyl-transferase Complex as potential regulator of
FXN repression
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.
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