Antisense oligonucleotides as a potential treatment for brain deficits observed in myotonic dystrophy type 1

Abstract

Myotonic dystrophy, or dystrophia myotonica type 1 (DM1), is a multi-systemic disorder and is the most common adult form of muscular dystrophy. It affects not only muscles but also many organs, including the brain. Cerebral impairments include cognitive deficits, daytime sleepiness, and loss of visuospatial and memory functions. The expression of mutated transcripts with CUG repeats results in a gain of toxic mRNA function. The antisense oligonucleotide (ASO) strategy to treat DM1 brain deficits is limited by the fact that ASOs do not cross the blood-brain barrier after systemic administration, indicating that other methods of delivery should be considered. ASO technology has emerged as a powerful tool for developing potential new therapies for a wide variety of human diseases, and its potential has been proven in a recent clinical trial. Targeting DMPK mRNA in neural cells derived from human induced pluripotent stem cells obtained from a DM1 patient with the IONIS 486178 ASO abolished CUG-expanded foci, enabled nuclear redistribution of MBNL1/2, and corrected aberrant splicing. Intracerebroventricular injection of the IONIS 486178 ASO in DMSXL mice decreased the levels of mutant DMPK mRNAs by up to 70% throughout different brain regions. It also reversed behavioral abnormalities following neonatal administration. The present study indicated that the IONIS 486178 ASO targets mutant DMPK mRNAs in the brain and strongly supports the feasibility of a therapy for DM1 patients based on the intrathecal injection of an ASO.

Fig. 1. DM1 NPC model for antisense oligonucleotide screening.

A Immunofluorescence of reprogrammed iPSC lines for the surface antigen TRA-1-60 and the nuclear pluripotency marker NANOG. B Loss of the pluripotency markers OCT4 and NANOG and expression of the neuroectodermal markers NES and PAX6. C Forebrain identity of neurons after 15 days of maturation. Patch-clamp measured D sodium currents, and E action potentials and cell-attached recordings of spontaneous cell firing activity. F Southern blot of DM1 iPSCs with a large repeat expansion in the DMPK gene. G FISH showing the presence of CUG-expanded foci in DM1 iPSCs and NPCs. H Quantification of overall CUG foci by FISH. Statistical analyses were performed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. I RT-qPCR DMPK mRNA analysis after a treatment with 500 nM IONIS 486178 ASO. A two-tailed unpaired Student’s t-test with Welch’s correction was used to determine the significance between the two groups. The error bars are presented as the mean ± SEM. SEM standard error of the mean.

Fig. 2. Correction of NPC DM1 phenotypes after a treatment with the IONIS 486178 ASO.

A FISH immunofluorescence showing the destruction of nuclear foci and the redistribution of MBNL1 and B MBNL2 24 h after transfection with 500 nM IONIS 486178 ASO. C RT-PCR of DM1 alternative mis-splicing. Statistical analyses were performed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. The error bars are presented as the mean ± SEM. Scale bar: 20 µm.

Fig. 3. Intracerebroventricular injection of the IONIS 486178 ASO in DMSXL mice.

RT-qPCR hDMPK mRNA analysis of a dose-dependent (A) and a time-dependent (B) ASO treatment in different brain areas. An ordinary two-way ANOVA with Tukey’s post hoc analysis for comparing multiple groups was used. The error bars are presented as the mean ± SD. SD standard deviation.

Fig. 4. Mouse brain distribution of the IONIS 486178 ASO.

Distribution of the ASO in the DMSXL mouse brain 1, 3, and 12 weeks after a 75 μg i.c.v. bolus injection. The ASO was visualized by immunostaining with an anti-ASO antibody (6651 Pan ASO) followed by counterstaining with hematoxylin. The scale bar of all images is shown on bottom right image.

Fig. 5. IONIS 486178 ASO toxicity profile in DMSXL mouse.

A Aif1 and Gfap mRNA expression profiles 1, 3, 12, and 24 weeks after a 75 μg i.c.v. bolus injection. Liver and kidney histology (B), the scale bar of all images is shown on bottom right image. Blood chemistry (C). ALP alkaline phosphatase, ALT alanine transaminase, AST aspartate transaminase, CK creatine kinase, CRE creatinine. Statistical analyses were performed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. The error bars are presented as the mean ± SD.

Fig. 6. Behavioral abnormality correction of DMSXL mice after an IONIS 486178 ASO treatment.

RT-qPCR hDMPK (A) and Aif1 and Gfap (B) mRNA analysis of the brain. Schematic zones in the open field arena (C) and representative traces of mouse movement (D). Total distance traveled (E), time mobile (F), and average speed (G) of DMSXL homozygote mice. Number of entries into the zone (H) and duration of visit in the zone (I) of the open field arena. The statistical analyses were performed using a standard two-tailed unpaired Student’s t-test for A and B, an ordinary one-way ANOVA with Tukey’s multiple comparisons test for F. a Brown–Forsythe ANOVA test with Games-Howell’s multiple comparisons test for E and G and an ordinary two-way ANOVA with Tukey’s multiple comparisons test for H and I. The error bars are presented as the mean ± SEM for A and B or ± SD for E–I.