Adding a temporal dimension to the study of Friedreich's ataxia: the effect of frataxin overexpression in a human cell model

Abstract

The neurodegenerative disease Friedreich's ataxia is caused by lower than normal levels of frataxin, an important protein involved in iron–sulfur (Fe-S) cluster biogenesis. An important step in designing strategies to treat this disease is to understand whether increasing the frataxin levels by gene therapy would simply be beneficial or detrimental, because previous studies, mostly based on animal models, have reported conflicting results. Here, we have exploited an inducible model, which we developed using the CRISPR/Cas9 methodology, to study the effects of frataxin overexpression in human cells and monitor how the system recovers after overexpression. Using new tools, which range from high-throughput microscopy to in cell infrared, we prove that overexpression of the frataxin gene affects the cellular metabolism. It also leads to a significant increase of oxidative stress and labile iron pool levels. These cellular alterations are similar to those observed when the gene is partly silenced, as occurs in Friedreich's ataxia patients. Our data suggest that the levels of frataxin must be tightly regulated and fine-tuned, with any imbalance leading to oxidative stress and toxicity.

Impact of Drosophila Models in the Study and Treatment of Friedreich’s Ataxia

“…Indeed, studies in Drosophila showed that overexpression of endogenous or human frataxin in the nervous system decreased longevity, affected locomotor activity, and induced neurodegeneration [ 37 , 38 ], revealing the importance of a finely controlled level of expression of frataxin in the development of gene therapy approaches. Very recently, a study using human cells confirmed the hypothesis from these fly reports [ 39 ].…”

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

“…43,44 In addition, uncontrolled expression may be of particular importance in the case of FRDA and other diseases where overexpression of the gene may lead to toxic side effects, potentially reducing the therapeutic benefit of the therapy. [26][27][28]30 Prior studies in Drosophila as well as more recent work in human cell models demonstrated that overexpression of frataxin affects cellular metabolism, increases oxidative stress and labile iron pool levels. [26][27][28]30 Considering that similar phenotypic effects are observed upon frataxin depletion, it reaffirms the notion that fine-tuning of frataxin levels is important for cellular fitness.…”

“…[26][27][28]30 Prior studies in Drosophila as well as more recent work in human cell models demonstrated that overexpression of frataxin affects cellular metabolism, increases oxidative stress and labile iron pool levels. [26][27][28]30 Considering that similar phenotypic effects are observed upon frataxin depletion, it reaffirms the notion that fine-tuning of frataxin levels is important for cellular fitness. On the contrary, initial proof-of-concept gene therapy studies demonstrated that significant overexpression of human FXN delivered by AAV in conditional mouse models of FRDA rescues severe phenotypes associated with complete loss of frataxin.…”

“…15,25 However, a growing number of reports indicate that an uncontrolled increase of frataxin levels significantly above physiological levels may lead to oxidative damage and cell death, which are changes similar to FXN downregulation. [26][27][28][29][30] This suggests that a certain cellular tolerance exists for fluctuations in FXN levels, but moving out of a tolerable range (in any direction) may have detrimental consequences. Thus, defining the endogenous promoter may facilitate proper regulation of FXN expression, especially in light of current gene therapy efforts.…”

A review of genetic engineering biotechnologies for enhanced chronic wound healing

“…The multiple functions of CRISPR-Cas9 are potentially very useful for chronic wounds, particularly given the success found in other disease states. 69,[71][72][73] First, CRISPR-Cas9 constructs are relatively simple to construct and are small. Second, CRISPR-Cas9 can specifically modify nearly any genomic target without off-target effects 74 and can use the gene library already found in the host genome rather than having to consistently rely on inserting exogenous DNA.…”