Development of frataxin gene expression measures for the evaluation of experimental treatments in Friedreich's ataxia

Heather L Plasterer, Eric C Deutsch, Matthew Belmonte, Elizabeth Egan, David R Lynch, James R Rusche, Heather L Plasterer, Eric C Deutsch, Matthew Belmonte, Elizabeth Egan, David R Lynch, James R Rusche

Abstract

Background: Friedreich ataxia is a progressive neurodegenerative disorder caused by GAA triplet repeat expansions or point mutations in the FXN gene and, ultimately, a deficiency in the levels of functional frataxin protein. Heterozygous carriers of the expansion express approximately 50% of normal frataxin levels yet manifest no clinical symptoms, suggesting that therapeutic approaches that increase frataxin may be effective even if frataxin is raised only to carrier levels. Small molecule HDAC inhibitor compounds increase frataxin mRNA and protein levels, and have beneficial effects in animal models of FRDA.

Methodology/principal findings: To gather data supporting the use of frataxin as a therapeutic biomarker of drug response we characterized the intra-individual stability of frataxin over time, determined the contribution of frataxin from different components of blood, compared frataxin measures in different cell compartments, and demonstrated that frataxin increases are achieved in peripheral blood mononuclear cells. Frataxin mRNA and protein levels were stable with repeated sampling over four and 15 weeks. In the 15-week study, the average CV was 15.6% for protein and 18% for mRNA. Highest levels of frataxin in blood were in erythrocytes. As erythrocytes are not useful for frataxin assessment in many clinical trial situations, we confirmed that PBMCs and buccal swabs have frataxin levels equivalent to those of whole blood. In addition, a dose-dependent increase in frataxin was observed when PBMCs isolated from patient blood were treated with HDACi. Finally, higher frataxin levels predicted less severe neurological dysfunction and were associated with slower rates of neurological change.

Conclusions/significance: Our data support the use of frataxin as a biomarker of drug effect. Frataxin levels are stable over time and as such a 1.5 to 2-fold change would be detectable over normal biological fluctuations. Additionally, our data support buccal cells or PBMCs as sources for measuring frataxin protein in therapeutic trials.

Conflict of interest statement

Competing Interests: DRL, ECD, MB, and EE have declared that no competing interests exist. JRR is an employees of Repligen and HLP is a consultant for Repligen. This corporate involvement does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Frataxin protein and mRNA level…
Figure 1. Frataxin protein and mRNA level is stable from day-to-day in whole blood.
(A) Frataxin protein levels measured by dipstick assay in FRDA patient subjects over the course of 4 weeks (Cohort 1; n = 31). (B) Frataxin protein levels measured by dipstick assay in whole blood collected from 5 FRDA patients (red) and 5 related carriers (blue) weekly for 15 weeks (Cohort 2). (C) Frataxin mRNA levels measured by RT-qPCR in blood collected in PAXgene tubes from Cohort 2. Frataxin mRNA level is expressed relative to control donor levels and is normalized to the endogenous control gene GAPDH.
Figure 2. Frataxin protein and mRNA levels…
Figure 2. Frataxin protein and mRNA levels strongly correlated when measured in different cellular compartments (Cohort 3).
(A) Frataxin mRNA in PBMCs and whole blood (R2 = 0.84, p<0.0001). (B) Frataxin protein in PBMCs and whole blood (R2 = 0.63, p<0.0001). (C) Frataxin mRNA and protein in whole blood (R2 = 0.55, p<0.0001). (D) Frataxin mRNA and protein in PBMCs (R2 = 0.58, p<0.0001). (E) Frataxin protein in PBMCs and cheek swabs (R2 = 0.67, p<0.0001). (F) Frataxin protein in whole blood and cheek swabs (R2 = 0.58, p<0.0001). Frataxin mRNA level in PBMCs and whole blood is expressed relative to control donor levels and is normalized to the endogenous control gene GAPDH.
Figure 3. Effect of HDAC inhibitor RG2833…
Figure 3. Effect of HDAC inhibitor RG2833 on frataxin level in PBMCs (Cohort 3).
(A) The effect of RG2833 on frataxin mRNA level relative to vehicle control in PBMCs following 48 hour treatment (n = 49 at 1, 2.5, 5, and 10 µM; n = 2 at 0.1 and 0.5 µM). The relative quantity of frataxin mRNA in HDACi–treated PBMCs is normalized to cell number and RNA input. (B) The effect of RG2833 on FXN protein level relative to vehicle control in PBMCs following 72 hour treatment (n = 27). (C) The fold-change increase in FXN mRNA in PBMC in response to HDACi does not correlate with repeat length (GAA1, R2 = 0.015, p = 0.451). (D) The fold-change increase in FXN protein in response to HDACi does not correlate with repeat length (GAA1, R2 = 0.006, p = 0.686).

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