Friedreich’s Ataxia (FRDA) is the most common autosomal recessive neuromuscular disorder. The disease is
caused by expanded GAA repeats in the first intron of the frataxin (FXN) gene. No effective treatments for the
disease are available, owing to the expanded repeats remaining in the patients’ genome. Thus, a treatment that
targets the expanded GAA repeats is urgently needed. We found that the inhibition of H3K9 trimethylation
(H3K9me3) synergized with DNA base excision repair (BER) to contract the expanded GAA repeats and
upregulate FXN gene expression in FRDA neural cells and transgenic mouse brain. We hypothesize that GAA
repeat-targeted demethylation of H3K9me2/me3 at the FXN gene can disrupt heterochromatin and induce BER
to contract the expanded repeats. To test this hypothesis, we propose to use a CRISPR/Cas9 system with the
histone H3-trimethyl-L-Lysine 9 demethylase 4D (KDM4D) fused to catalytically inactivated S. pyogenes Cas9
(CRISPR/dCas9-KDM4D) to induce GAA repeat-targeted demethylation of H3K9me2/me3 in FRDA neural cells.
We will pursue two Specific Aims. Aim 1 is to determine if the GAA repeat-targeted CRISPR/dCas9-KDM4D can
demethylate H3K9me2/me3 to disrupt heterochromatin at the FXN gene in FRDA neural cells. First, we will fuse
the human KDM4D gene with the S. pyogenes dCas9 using the plasmid pCRISPR/dCas9-DNMT3A-PuroR_v2
as a backbone. KDM4D will be linked to the C-terminus of dCas9 through the XTEN80 linker chain. The
sequences for coding the single-strand guide RNAs (sgRNAs) that target the 5’- or 3’-flanking regions of the
expanded GAA repeats will also be inserted into the plasmid. The plasmid will be stably transfected into FRDA
neural progenitor cells (NPCs) differentiated from induced pluripotent stem cells (iPSCs) of an FRDA patient.
Second, we will determine if the repeat-targeted dCas9-KDM4D can reduce the level of H3K9me2/me3 and
alleviate heterochromatinization on the expanded repeats in FRDA neural cells differentiated from NPCs. Aim 2
is to determine if the GAA repeat-targeted CRISPR/dCas9-KDM4D promotes GAA repeat contraction through
BER, leading to the upregulation of the FXN gene expression and the alleviation of mitochondrial dysfunction in
FRDA neural cells. First, we will determine if dCas9-KDM4D can lead to GAA repeat contraction. We will then
determine if dCas9-KDM4D can facilitate the recruitment of the key BER enzymes, DNA polymerase ß (Pol ß),
and flap endonuclease 1 (FEN1) to the expanded repeats in FRDA neural cells. Second, we will test if dCas9-
KDM4D can result in the upregulation of the FXN gene expression and alleviate mitochondrial dysfunction. Our
study will provide proof of concept for a gene-targeted contraction of expanded GAA repeats via the synergy
between histone modifications and DNA repair. The results will reveal the mechanisms underlying
CRISPR/dCas9-KDM4D targeted contractions of expanded GAA repeats through the interplay of histone
demethylation with BER. The study will further open a new avenue to develop effective gene therapy for FRDA.
