Friedreich’s ataxia (FRDA) is the most common autosomal recessive ataxia that occurs in the white population.
The disease is caused by expanded GAA repeats at the first intron of the frataxin (FXN) gene. There is no cure
for the disease due to the inherited expanded GAA repeats in the FRDA patient’s genome. Thus, it is urgent to
develop FXN gene-targeted GAA repeat contraction for FRDA treatment. Recent studies from our group have
found that inhibition of histone H3 lysine 9 (H3K9) methylation can lead to large GAA repeat contraction in FRDA
neural cells and transgenic mouse brain by opening the chromatin and promoting DNA base excision repair
(BER) at the FXN gene. We further hypothesize that FXN gene-targeted histone demethylation interplays with
BER to contract GAA repeats and activate the FXN gene in FRDA. The hypothesis will be tested by Dr. Rhyisa
Armbrister, the postdoctoral fellow of Dr. Yuan Liu, the PI of the NIHR03 project. The project will be conducted
by employing novel human natural vault nanoparticles encapsulated with CRISPR/dCas9-H3K9 demethylase,
KDM4D-sgRNA complex, inducing FXN gene-targeted histone demethylation and BER-mediated contraction of
the expanded GAA repeats in differentiated FRDA neural cells. The goal will be accomplished by pursuing two
Specific Aims under Dr. Liu’s supervision and mentorship through collaboration with Dr. Cheng-Yu Lai. Aim 1 is
to determine if vault nanoparticles encapsulated with FXN gene-targeted CRISPR/dCas9-KDM4D can lead to
the histone demethylation and contraction of the expanded GAA repeats in FRDA neural cells. We will generate
recombinant human vault nanoparticles encapsulated with dCas9-KDM4D fusion proteins assembled with
synthesized FXN gene-targeted sgRNAs and transduce vault nanoparticles into differentiated FRDA neural cells.
We will then determine if vault nanoparticle-mediated FXN gene-targeted histone demethylation can result in the
contraction of the expanded GAA repeats in FRDA neural cells. Aim 2 is to determine if vault nanoparticle-
mediated FXN gene-targeted histone demethylation can disrupt heterochromatinization to induce BER on the
expanded GAA repeats, thereby leading to upregulation of FXN gene expression and relief of FRDA
neurodegenerative phenotypes. We will determine if vault-mediated FXN gene-targeted H3K9 demethylation
can reduce heterochromatin protein 1α and 1β, increase H3K9ac, and induce the recruitment of DNA base
excision repair enzymes on the expanded repeats, leading to FXN gene upregulation in FRDA neural cells and
relief of FRDA phenotypes. Our study will integrate the CRISPR/dCas9 system with human vault
bionanoparticles to reveal the novel mechanisms of FXN gene-targeted GAA repeat contraction via histone
demethylation and DNA repair. The study will create the first platform for bionanoparticle-mediated gene therapy,
forging a new avenue for innovative treatments for FRDA and other repeat expansion diseases. Furthermore,
the project will provide an excellent opportunity for Dr. Armbrister to develop her future career as a tenure-track
faculty in neuroscience, thereby promoting underrepresented minorities and diversity in neuroscience.
