CRISPR/dCas9-Targeted Histone Demethylation Interplays with DNA Repair to Contract GAA Repeats in Friedreich's Ataxia - Friedreich’s ataxia (FRDA) is the most common autosomal recessive ataxia. It is caused by expanded GAA repeats at the first intron of the frataxin (FXN) gene. No effective treatments are available for the disease due to the inherited expanded GAA repeats in the patient’s genome. Thus, there is an urgent need to develop FXN gene-targeted GAA repeat contraction for FRDA treatment. We have recently found that inhibition of histone H3 lysine 9 (H3K9) methylation can induce large GAA repeat contraction in FRDA neural cells and transgenic mice by opening the chromatin and promoting DNA base excision repair (BER) at the FXN gene. We hypothesize that FXN gene-targeted histone demethylation interplays with BER to contract GAA repeats and activate the FXN gene in FRDA. We will test the hypothesis using CRISPR/deadCas9 (dCas9)-mediated FXN gene-targeted histone demethylation to disrupt heterochromatin, induce BER-mediated GAA repeat contraction, activate FXN gene and relieve FRDA phenotypes. This will be achieved by the integrated indispensable expertise of all the PIs through their synergistic team efforts. First, we will determine if CRISPR/dCas9-mediated FXN gene- targeted histone demethylation can alleviate heterochromatinization and induce BER, leading to GAA repeat contraction and relief of FRDA phenotypes. We will determine if FXN gene-targeted histone demethylases KDM4D, KDM6A, and KDM6B can induce BER to contract GAA repeats, relieving FRDA phenotypes in FRDA neural and cardiac cells. Second, we will determine if the human vault nanoparticle-mediated FXN gene-targeted histone demethylation can lead to GAA repeat contraction and relief of FRDA phenotypes. We will encapsulate sgRNA-dCas9-histone demethylases into recombinant vault nanoparticles. We will then test if the vault-mediated histone demethylation can induce GAA repeat contraction via BER, activate the FXN gene, alleviate FRDA phenotypes, and interplay with the function of the blood-brain barrier. Third, we will determine if FXN gene- targeted histone demethylation can lead to cellular differential effects on GAA repeat contraction, FXN gene activation, and relief of FRDA phenotypes via cell-cell interaction. Using a single-cell analysis, we will determine if FXN gene-targeted histone demethylation can cause cellular differential effects on GAA repeat contraction, FXN gene activation, and energy production via cell-cell communication. Fourth, we will determine if FXN gene- targeted dCas9-histone demethylases can coordinate with a nucleosome to demethylate histones on expanded GAA repeats at the FXN gene. We will use molecular dynamics simulation, machine learning, and the AI tool AlphaFold3 to determine the FXN gene-targeted substrate recognition of dCas9-histone demethylases through their interaction with a nucleosome. Our study will reveal the novel mechanisms of FXN gene-targeted GAA repeat contraction, create a bionanoparticle-mediated FXN gene-targeted platform for GAA repeat contraction, and open a new horizon for developing AI-assisted mechanism-based gene therapy for FRDA. Thus, it will fundamentally transform the research and treatment for FRDA and other neurological diseases.
