Anti-gene oligonucleotides targeting Friedreich's ataxia expanded GAA⋅TTC repeats increase Frataxin expression

Abstract

Friedreich's ataxia is a progressive, autosomal recessive ataxia caused, in most cases, by homozygous expansion of GAA⋅TTC triplet-repeats in the first intron of the Frataxin gene. GAA⋅TTC repeat expansion results in the formation of a non-B-DNA intramolecular triplex as well as changes in the epigenetic landscape at the Frataxin locus. Expansion of intronic GAA⋅TTC repeats is associated with reduced levels of Frataxin mRNA and protein, resulting in disease development. In our previous study, we demonstrated that DNA-binding anti-gene oligonucleotides specifically targeting the GAA⋅TTC repeat expansion effectively disrupted the formation of intramolecular triplex structures. In this study, we extend these findings by showing that targeting repeat-expanded chromosomal DNA with anti-gene oligonucleotides leads to an increase in Frataxin mRNA and protein levels in cells derived from Friedreich's ataxia patients. We examined numerous anti-gene oligonucleotides and found that the design, length, and their locked nucleic acid composition have a high impact on the effectiveness of the treatment. Collectively, our results demonstrate the unique capability of specifically designed oligonucleotides targeting the GAA⋅TTC DNA repeats to upregulate Frataxin gene expression.

Keywords: DNA targeting; Friedreich’s ataxia; H-DNA; Huntingtonś disease; MT: Oligonucleotides: Therapies and Applications; anti-gene; frataxin; oligonucleotide; tandem repeats; therapeutic oligonucleotide optimization; trinucleotide repeat expansions.

Graphical abstract

Figure 1

LNA composition influences the effect of GAA and CTT ONs on FXN mRNA expression (A) Schematic representation of GAA and CTT ON interactions with the FXN gene and their proposed outcomes. (B) Illustrations of different GAA and CTT ONs used in this study. LNA bases are darker with white text, and DNA bases are in a lighter color with black text. (C) Chemical structures of the nucleic acid modifications used in this study. (D) Female FRDA patient-derived fibroblasts carrying 330/380 GAA⋅TTC repeats (GM03816) were treated with 200 nM GAA, CTT and IRL ONs, and FXN mRNA levels were analyzed with RT-qPCR 4 days after transfection. Relative FXN expression of each treatment was compared with nontreated (NT) cells and normalized to the ratio of the FXN gene to HPRT1. Results are presented as mean ± SD (n = 4). Statistical analysis was performed using one-way ANOVA, multiple comparisons (Dunnett) (∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns = nonsignificant). (A), (B), and (C) were created in BioRender by M.N. (2025), https://BioRender.com/e45v370.

Figure 2

FXN upregulation improves with increasing GAA A-GO length at 200 nM transfection, but not at 3 μM gymnotic delivery Female FRDA patient-derived fibroblasts carrying 330/380 GAA⋅TTC repeats (GM03816) were treated with (A) 200 nM GAA, CTT and IRL ONs, and FXN mRNA levels were analyzed with RT-qPCR 4 days after transfection. Relative FXN expression of each treatment was compared with NT cells and normalized to the ratio of the FXN gene to HPRT1. Results are presented as mean ± SD (n = 3). Statistics were performed with one-way ANOVA toward the corresponding random IRL ON consisting of the same LNA numbers and constitutions (IRL ₁₅₍₁₎). (B) 3 μM ONs in medium supplemented with 9 mM CaCl₂ were added to the cells the day after plating. After 4 days, total RNA was extracted, mRNA was quantified with RT-qPCR, and FXN levels were normalized to HPRT1 and compared with NT cells. Results are presented as mean with SD (n ≥ 3). Statistics were performed with one-way ANOVA multiple comparisons (Šidák) toward corresponding controls (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 3

Dose-dependent FXN mRNA expression after gymnotic delivery of ONs Female FRDA patient-derived fibroblasts carrying 330/380 GAA⋅TTC repeats (GM03816) (A–C) were treated with ONs at concentrations ranging from 0.18 to 6 μM and (D) 4869 fibroblasts were treated with selected concentrations of 0.37 and 0.75 μM, both in a medium supplemented with 9 mM CaCl₂. Four days post-treatment the cells were harvested, and FXN mRNA levels were analyzed. The values were normalized to HPRT1 as a reference gene and compared with NT cells. Results are presented as mean ± SD (n ≥ 3). Statistics for sections (A), (B), and (D) were performed with one-way ANOVA Multiple Comparison (Šidák) toward the corresponding concentration of IRL ON (∗p < 0.05, ∗∗p < 0.01). Statistical analysis for (C) was performed using the Kruskal-Wallis test. IRL ONs are randomly 15- or 24-nt-long ONs that are not complementary to any related genes in this project.

Figure 4

Dose-response of gymnotically-delivered ONs on the FXN mRNA expression GM03816 cells were treated with (A) 16-mer and (B) 19-mer ONs at concentrations ranging from 0.18 to 6 μM in medium supplemented with 9 mM CaCl₂. After 4 days of treatment, the cells were harvested, and FXN mRNA expression levels were analyzed. The values were normalized to HPRT1 levels as the reference gene and were compared to NT cells. Results are presented as mean ± SD. Statistics were performed with one-way ANOVA multiple comparisons (Šidák), toward the IRL control (∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 5

GAA A-GOs increase the FXN protein levels (A) Increased effect of ONs with 40% of LNA on FXN protein expression 4 days post-transfection with 100 nM ONs in GM03816 cells using Lipofectamine LTX. The FXN protein was normalized to Actin levels as a reference gene and relative FXN expression is shown after normalizing to the control NT cells. Results are presented as mean ± SD (n ≥ 3). (B) Representative western blot of the treatment presented in (A). (C) The FXN protein levels were measured by western blotting 4 days after the gymnotic delivery of GAA₂₄, CTT₂₄, and IRL₁₅₍₂₎ in the presence of a medium supplemented with 9 mM CaCl₂. IRL ON (IRL₍₁₅₎₂) is random 15 nt long ON that is not complementary to any related genes in this project and the LNA composition is the same as the GAA and CTT ONs. The values were normalized to Actin levels as a reference gene and compared with NT cells. Results are presented as mean ± SD (n = 3). Statistics were performed with one-way ANOVA multiple comparisons (A: Šidák and B: Fisher’s least significant difference test), toward corresponding controls. (D) Representative western blot of the treatment presented with GAA₂₄, CTT₂₄, and IRL₁₅₍₂₎ in medium supplemented with 9 mM CaCl₂ collected 4 days after treatments.

Figure 6

Increasing the GAA A-GO length enhances FXN upregulation in a FRDA cell model with a higher number of GAA⋅TTC repeats GM03665 fibroblasts were treated with ONs of different lengths of GAA, CTT, and IRL at 200 nM. Treated and nontreated cells were harvested 4 days after transfection, and FXN mRNA levels were assessed by RT-qPCR. The values were normalized to HPRT1 and were compared with NT cells. Results are presented as mean ± SD (n ≥ 3). Statistics were performed with one-way ANOVA Multiple Comparison (Šidák), toward control ONs (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 7

Dose-dependent effect of gymnotically-delivered GAA₂₄ on FXN expression FRDA fibroblasts containing a higher number of GAA⋅TTC repeats (GM03665) were treated with GAA₂₄ and CTT₂₄ ONs at concentrations ranging from 0.18 to 3 μM in medium supplemented with 9 mM CaCl₂. For IRL₂₄, only selected concentrations were used. Treated and NT cells were harvested 4 days post-treatments, and FXN mRNA levels were assessed by RT-qPCR. The values were normalized to HPRT1 levels as reference gene and were compared with NT cells. Results are presented as mean ± SD (n ≥ 3). Statistics were performed with one-way ANOVA multiple comparisons (Holm- Šidák) toward control ONs (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).