shRNA Molecules Reverse Friedreich’s Ataxia in Cells

A screen of molecules known as short-hairpin-loop-RNA (shRNA) revealed that certain features of Friedreich’s ataxia can be relieved through two specific shRNA sequences. In the laboratory of Dr. Robert B. Wilson at University of Pennsylvania School of Medicine, researchers conducted a study that exposed cells isolated from Friedreich’s ataxia patients to shRNAs and rescued the cells’ expression of frataxin.

Frataxin deficiency, the root of Friedreich’s ataxia, is detrimental to mitochondrial function. The protein is a chaperone to iron-sulfur clusters that enable the respiratory complexes required for cells to turn glucose into energy during oxidative phosphorylation inside the mitochondria. As such, cells from Friedreich’s ataxia patients are particularly sensitive to beta-hydroxybutyrate (BHB) because this molecule interferes further with glucose metabolism.

“We took advantage of the particular sensitivity of these cells to BHB-based media to identify shRNAs that reverse this sensitivity,” wrote lead author, Dr. M. Grazia Cotticelli, in the article, “Phenotypic Screening for Friedreich Ataxia Using Random shRNA Selection,” which was published in Journal of Biomolecular Screening.

Dr. Cotticelli, Dr. Wilson, and the rest of the research team first started with a library of shRNA molecules they had discovered in a previous study. The library contains approximately 300,000 completely random shRNA molecules, allowing an unbiased screen for ones that may be effective. The researchers applied these shRNA sequences to fibroblast cells from patients and analyzed the cells that remained alive following exposure to BHB.

Two shRNA molecules successfully enhanced the growth of cells in BHB media: clone gFA2 and clone gFA11. Only gFA2 increased the expression of frataxin protein. Cells treated with gFA2 saw a doubling of frataxin mRNA.

“One of the shRNA sequences we identified increases frataxin expression,” wrote the authors, “thereby partially reversing the primary defect in Friedreich’s ataxia.” Since shRNA integrates into a cell’s genome permanently, developing a shRNA-based treatment similar to gFA2 may benefit Friedreich’s ataxia patients in both the short- and the long-term. Strategies such as these are essential for helping patients, as there are currently no adequate treatments for the condition.