Excision of the expanded GAA repeats corrects cardiomyopathy phenotypes of iPSC-derived Friedreich's ataxia cardiomyocytes

Li, Jixue; Rozwadowska, Natalia; Clark, Amanda D.; Fil, Daniel; Napierala, Marek

doi:10.1016/j.scr.2019.101529

Cited by 29 publications

(37 citation statements)

References 57 publications

(93 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To assess whether the expression levels of FXN, BDNF, and miRNA-10a-5p are interdependent, we edited the expanded GAA repeats from FRDA fibroblasts using ZFNs. Previously, we demonstrated that heterozygous excision of the GAA repeats (removal of one of the expanded GAA tracts) increases FXN expression and alleviates some of the phenotypic changes characteristic of FRDA neurons and cardiomyocytes differentiated from induced pluripotent stem cells [33,41]. To further increase FXN expression to a level comparable to that of unaffected cells, we excised a second expanded GAA tract using the same pair of ZFNs as described previously in [33] (Fig.…”

Section: Excision Of Gaa Repeats Increases Fxn and Bdnf Expression Whmentioning

confidence: 99%

A Comprehensive Transcriptome Analysis Identifies FXN and BDNF as Novel Targets of miRNAs in Friedreich’s Ataxia Patients

et al. 2020

Self Cite

View full text Add to dashboard Cite

Friedreich's ataxia (FRDA) is a genetic neurodegenerative disease that is caused by guanine-adenine-adenine (GAA) nucleotide repeat expansions in the first intron of the frataxin (FXN) gene. Although present in the intron, this mutation leads to a substantial decrease in protein expression. Currently, no effective treatment is available for FRDA, and, in addition to FXN, other targets with therapeutic potential are continuously sought. As miRNAs can regulate the expression of a broad spectrum of genes, are used as biomarkers, and can serve as therapeutic tools, we decided to identify and characterize differentially expressed miRNAs and their targets in FRDA cells compared to unaffected control (CTRL) cells. In this study, we performed an integrated miRNAseq and RNAseq analysis using the same cohort of primary FRDA and CTRL cells. The results of the transcriptome studies were supported by bioinformatic analyses and validated by qRT-PCR. miRNA interactions with target genes were assessed by luciferase assays, qRT-PCR, and immunoblotting. In silico analysis identified the FXN transcript as a target of five miRNAs upregulated in FRDA cells. Further studies confirmed that miRNA-224-5p indeed targets FXN, resulting in decreases in mRNA and protein levels. We also validated the ability of miRNA-10a-5p to bind and regulate the levels of brain-derived neurotrophic factor (BDNF), an important modulator of neuronal growth. We observed a significant decrease in the levels of miRNA-10a-5p and increase in the levels of BDNF upon correction of FRDA cells via zinc-finger nuclease (ZFN)-mediated excision of expanded GAA repeats. Our comprehensive transcriptome analyses identified miRNA-224-5p and miRNA-10a-5p as negative regulators of the FXN and BDNF expression, respectively. These results emphasize not only the importance of miRNAs in the pathogenesis of FRDA but also their potential as therapeutic targets for this disease.

show abstract

Section: Excision Of Gaa Repeats Increases Fxn and Bdnf Expression Whmentioning

confidence: 99%

A Comprehensive Transcriptome Analysis Identifies FXN and BDNF as Novel Targets of miRNAs in Friedreich’s Ataxia Patients

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recent studies demonstrated that excision of the tract, together with flanking sequences, using editing nucleases, increases FXN expression to near unaffected control levels. [31][32][33] This strongly suggests that no critical regulatory elements exist in the direct vicinity of the GAAs. However, intron 1 of the FXN gene encodes numerous interspersed repeat elements making genome editing of this region a challenging task.…”

Section: Discussionmentioning

confidence: 99%

“…The iPSCs were cultured using mTeSR-1 (StemCell Technologies) as we described. 32 Cells were differentiated into cardiomyocytes using the Gibco Ò PSC Cardiomyocyte Differentiation Kit (Gibco, A2921201) according to the manufacturer's recommendations. Neuronal cells were obtained via Ngn2 overexpression-mediated differentiation according to Ref.…”

Section: Culturing Ipscs Neuronal and Cardiac Differentiationmentioning

confidence: 99%

“…In addition, excision of the intronic expanded GAAs has been demonstrated in proof-of-concept studies to be an efficient, permanent correction of the genetic defect underlying FRDA. [31][32][33] Uncovering a potential regulatory element within intron 1 is of critical importance for judicial selection of cleavage sites for designer nucleases targeting the expanded GAAs.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy

Wang

et al. 2020

Human Gene Therapy

Self Cite

View full text Add to dashboard Cite

Friedreich's ataxia (FRDA) is the most common inherited form of ataxia in humans. It is caused by severe downregulation of frataxin (FXN) expression instigated by hyperexpansion of the GAA repeats located in intron 1 of the FXN gene. Despite numerous studies focused on identifying compounds capable of stimulating FXN expression, current knowledge regarding cis-regulatory elements involved in FXN gene expression is lacking. Using a combination of episomal and genome-integrated constructs, we defined a minimal endogenous promoter sequence required to efficiently drive FXN expression in human cells. We generated 19 constructs varying in length of the DNA sequences upstream and downstream of the ATG start codon. Using transient transfection, we evaluated the capability of these constructs to drive FXN expression. These analyses allowed us to identify a region of the gene indispensable for FXN expression. Subsequently, selected constructs containing the FXN expression control regions of varying lengths were site specifically integrated into the genome of HEK293T and human-induced pluripotent stem cells (iPSCs). FXN expression was detected in iPSCs and persisted after differentiation to neuronal and cardiac cells, indicating lineage independent function of defined regulatory DNA sequences. Finally, based on these results, we generated AAV encoding miniFXN genes and demonstrated in vivo FXN expression in mice. Results of these studies identified FXN sequences necessary to express FXN in human and mouse cells and provided rationale for potential use of endogenous FXN sequence in gene therapy strategies for FRDA.

show abstract

“…Several therapeutic approaches to arrest and/or slow down the disease are under development and can be grouped into those aimed at improving mitochondrial function and reducing oxidative stress, those trying to increase or stabilize frataxin levels, and gene therapy (for a review of FRDA therapeutic approaches, see [ 12 ]). Other emerging and promising therapies include stem cell therapy [ 13 ], genome editing [ 14 ], and oligonucleotide-based approaches [ 15 ]. As FRDA is a monogenic loss-of-function disease, it is an ideal candidate for gene therapy, as introducing a healthy copy of the gene is predicted to rescue the disease phenotype [ 16 , 17 ].…”

Section: Introductionmentioning

confidence: 99%

Altered Secretome and ROS Production in Olfactory Mucosa Stem Cells Derived from Friedreich’s Ataxia Patients

Pérez-Luz

Loría

Katsu-Jiménez

et al. 2020

IJMS

View full text Add to dashboard Cite

Friedreich’s ataxia is the most common hereditary ataxia for which there is no cure or approved treatment at present. However, therapeutic developments based on the understanding of pathological mechanisms underlying the disease have advanced considerably, with the implementation of cellular models that mimic the disease playing a crucial role. Human olfactory ecto-mesenchymal stem cells represent a novel model that could prove useful due to their accessibility and neurogenic capacity. Here, we isolated and cultured these stem cells from Friedreich´s ataxia patients and healthy donors, characterizing their phenotype and describing disease-specific features such as reduced cell viability, impaired aconitase activity, increased ROS production and the release of cytokines involved in neuroinflammation. Importantly, we observed a positive effect on patient-derived cells, when frataxin levels were restored, confirming the utility of this in vitro model to study the disease. This model will improve our understanding of Friedreich´s ataxia pathogenesis and will help in developing rationally designed therapeutic strategies.

show abstract

Excision of the expanded GAA repeats corrects cardiomyopathy phenotypes of iPSC-derived Friedreich's ataxia cardiomyocytes

Cited by 29 publications

References 57 publications

A Comprehensive Transcriptome Analysis Identifies FXN and BDNF as Novel Targets of miRNAs in Friedreich’s Ataxia Patients

A Comprehensive Transcriptome Analysis Identifies FXN and BDNF as Novel Targets of miRNAs in Friedreich’s Ataxia Patients

Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy

Altered Secretome and ROS Production in Olfactory Mucosa Stem Cells Derived from Friedreich’s Ataxia Patients

Contact Info

Product

Resources

About