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Friedreich ataxia (FRDA) is a multisystemic, autosomal recessive disorder caused by homozygous GAA expansion mutation in the first intron of frataxin (FXN) gene. FXN is a mitochondrial protein critical for iron-sulfur cluster biosynthesis and deficiency impairs mitochondrial electron transport chain functions and iron homeostasis within the organelle. Currently, there is no effective treatment for FRDA. We have previously demonstrated that single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in prevention of neurologic and cardiac complications of FRDA in YG8R mice, and rescue was mediated by FXN transfer from tissue engrafted, HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation, we developed an autologous stem cell transplantation approach using CRISPR/Cas9 for the excision of the GAA repeats in FRDA patients’ CD34+ HSPCs; this strategy leading to increased FXN expression and improved mitochondrial functions. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. We generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. iPSC derived FRDA neurons displayed characteristic apoptotic and mitochondrial phenotype of the disease, such as non-homogenous microtubule staining in neurites, increased caspase-3 expression, mitochondrial superoxide levels, mitochondrial fragmentation, and partial degradation of the cristae compared to healthy controls. These defects were fully prevented in the gene edited neurons. RNASeq analysis of FRDA and gene edited neurons demonstrated striking improvement in gene clusters associated with endoplasmic reticulum (ER) stress in the isogenic lines. Gene edited neurons demonstrated improved ER-calcium release, normalization of ER stress response gene, XBP-1, and significantly increased ER-mitochondrial contacts that are critical for functional homeostasis of both organelles, as compared to FRDA neurons. Ultrastructural analysis for these contact sites displayed severe ER structural damage in FRDA neurons, that was undetected in gene edited neurons. Taken together, these results represent a novel finding for disease pathogenesis showing dramatic ER structural damage in FRDA, validate the efficacy profile of our FXN gene editing approach in a disease relevant model, and support our approach as an effective strategy for therapeutic intervention for Friedreich’s ataxia.

Abstract Background RelA/p65 a crucial member of NF-κB signaling pathway plays diverse role in mediating oncogenesis. Limited knowledge prevails on the mechanistic insights of RelA gene regulation. RNA helicase p68 apart from being a vital player of RNA metabolism acts as a transcriptional coactivator of several oncogenic transcription factors including β-catenin and is highly implicated in cancer progression. In this study, we aim to discern the molecular mechanism of how an RNA helicase, p68 deploys a major oncogenic signaling pathway, Wnt/ β-catenin to regulate the expression of RelA, an indispensable component of NF-κB signaling pathway towards driving colon carcinogenesis. Methods Immunoblotting and quantitative RT-PCR was performed for determining the protein and mRNA expressions of the concerned genes respectively. Luciferase assay was employed for studying the promoter activity of RelA. Chromatin immunoprecipitation was used to evaluate the occupancy of transcription factors on the RelA promoter. Immunohistochemical analysis was conducted using FFPE sections derived from normal human colon and colon cancer patient samples. Finally, a syngeneic colorectal allograft mouse model was used to assess physiological significance of the in vitro findings. Results p68, β-catenin and RelA proteins were found to bear strong positive correlation in normal and colon carcinoma patient samples. Both p68 and β-catenin increased RelA mRNA and protein expression. p68, β-catenin and Wnt3a elevated RelA promoter activity. Conversely, p68 and β-catenin knockdown diminished RelA promoter activity and led to reduced RelA mRNA and protein expression. p68 was perceived to occupy RelA promoter with β-catenin at the TCF4/LEF (TBE) sites thereby potentiating RelA transcription. p68 and β-catenin alliance positively modulated the expression of signature NF-κB target genes. Enhanced NF-κB target gene expression by p68 was corroborated by findings in clinical samples. Tumors generated in mice colorectal allograft model, stably expressing p68 further reinforced our in vitro findings. Conclusions We report for the first time a novel mechanism of alliance between p68 and β-catenin in regulating the expression of RelA and stimulating the NF-κB signaling axis towards driving colon carcinogenesis. This study unravels novel modes of p68-mediated colon carcinogenesis, marking it a potential target for therapy.