Stabilization of expanded (CTG)•(CAG) repeats by antisense oligonucleotides

Abstract

Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the gene DMPK. The expansion is highly unstable in somatic cells, a feature that may contribute to disease progression. The RNA expressed from the mutant allele exerts a toxic gain of function, due to the presence of an expanded CUG repeat (CUG(exp)). This RNA dominant mechanism is amenable to therapeutic intervention with antisense oligonucleotides (ASOs). For example, CAG-repeat ASOs that bind CUG(exp) RNA are beneficial in DM1 models by altering the protein interactions or metabolism of the toxic RNA. Because CUG(exp) RNA has been shown to aggravate instability of expanded CTG repeats, we studied whether CAG-repeat ASOs may also affect this aspect of DM1. In human cells the instability of (CTG)(800) was suppressed by addition of CAG-repeat ASOs to the culture media. In mice that carry a DMPK transgene the somatic instability of (CTG)(800) was suppressed by direct injection of CAG-repeat ASOs into muscle tissue. These results raise the possibility that early intervention with ASOs to reduce RNA or protein toxicity may have the additional benefit of stabilizing CTG:CAG repeats at subpathogenic lengths.

Figure 1

Antisense oligonucleotides (ASOs) reduce RNA foci in HT1080 cells. (a) Diagram of pLC15-F construct for expression of expanded-CUG repeats in the DMPK 3′ UTR. The attB site supports genomic integration by PhiC31 integrase. CMV/CBA, CMV enhancer/chicken β-actin promoter; puro, puromycin resistance. (b) Sequence of LNA-ASOs. The position of locked nucleic acid (LNA)-modified subunits is shown by upper case letters, lower case letters are unmodified DNA. Gapmer and mixmer ASOs have full phosphorothioate backbones. (c) FISH showing foci of CUG^exp RNA (red) in nuclei (blue) of stably transfected HT1080 cells. (d) Histogram showing the percentage of cells with nuclear foci of CUG^exp RNA. The number of cells counted was 526 for no treatment, 300 for gapmer-treatment, and 398 for blocker-treatment. Mean ± SD, n = 3 or more. *P < 0.001. ASO, antisense oligonucleotide; CMV, cytomegalovirus.

Figure 2

RNA levels in CUG^exp-expressing or nontransfected HT1080 cells, determined by quantitative real-time reverse transcriptase (qRT)-PCR and normalized to 18S rRNA. (a) The level of DMPK 3′ UTR RNA (transgene + endogenous) was reduced by both antisense oligonucleotides (ASOs), to levels similar to nontransfected cells. (b) No effect of ASOs on expression of CASK, an endogenous transcript with (CUG)₁₆ in the 3′ UTR. (c) The level of DMPK 3′ UTR (transgene only) in cytoplasmic RNA was reduced by both ASOs. (d) No effect of ASOs on puro, expressed from the contiguous promoter. Data are the mean ± SD of triplicates. *P < 0.001.

Figure 3

Effects of CAG-repeat antisense oligonucleotide (ASO) on CTG•CAG repeat instability in HT1080 cells. (a) Representative data showing small-pool PCR followed by Southern blot for analysis of CTG repeat length. The scale on the left shows molecular weight markers converted into repeat number for CTG-repeat fragments of equivalent size. (b) Histograms showing repeat length distributions in HT1080 cells. The frequency distribution of unstable alleles is shown by gray bars (left vertical axis). The frequency of stable alleles is shown by black bars (right vertical axis). Allele lengths are grouped in bins spanning 50 repeats. More than 110 alleles were sized for each group.

Figure 4

Reduced R-loop formation by antisense oligonucleotides (ASOs) in HT1080 cells. (a) Detection of unpaired DNA at the 5′ proximal region of the CTG•CAG repeat tract. Shown are the positions of bisulfite-modified unpaired Cs on the nontemplate strand of representative clones. Vertical marks indicate C residues that have been converted to U. Numbers at the top of figure represent increments of 10 CTG repeats. (b) Effects of antisense oligonucleotides (ASOs) treatment on the frequency of C to U conversion in the CTG•CAG repeat. A total of 36 clones (corresponding to 6,468 CTG·CAG repeats) from nontemplate (CTG) strand and 30 clones (corresponding to 4,717 CTG·CAG repeats) from template (CAG) strand were analyzed. Data are indicated as the mean ± SE, n = 10 or more. *P < 0.01.

Figure 5

Effects of CAG-repeat antisense oligonucleotides (ASOs) on CTG•CAG repeat instability in XXL transgenic mice. (a) Small-pool PCR followed by Southern blot for analysis of CTG repeat length. The scale on the left shows molecular weight markers converted into numbers of CTG repeats. M indicates DIG-labeled molecular weight marker. (b) The percentage of unstable alleles in tibialis anterior muscle of XXL mice 4 weeks after the injection and electroporation of phosphate-buffered saline (PBS) or locked nucleic acid (LNA)-ASOs. Data from PBS vs. ASO-injected muscle from same mouse are connected by a solid or dotted line. (c) Level of human DMPK transgene mRNA determined by quantitative real-time reverse transcriptase (qRT)-PCR and normalized to 18S rRNA, expressed as the ratio of LNA-treated vs. PBS-treated TA muscles. Data are indicated as the mean ± SE of triplicates (n = 3 in each group). *P < 0.05.