Shorter Phosphorodiamidate Morpholino Splice-Switching Oligonucleotides May Increase Exon-Skipping Efficacy in DMD

Abstract

Duchenne muscular dystrophy is a fatal muscle disease, caused by mutations in DMD, leading to loss of dystrophin expression. Phosphorodiamidate morpholino splice-switching oligonucleotides (PMO-SSOs) have been used to elicit the restoration of a partially functional truncated dystrophin by excluding disruptive exons from the DMD messenger. The 30-mer PMO eteplirsen (EXONDYS51) developed for exon 51 skipping is the first dystrophin-restoring, conditionally FDA-approved drug in history. Clinical trials had shown a dose-dependent variable and patchy dystrophin restoration. The main obstacle for efficient dystrophin restoration is the inadequate uptake of PMOs into skeletal muscle fibers at low doses. The excessive cost of longer PMOs has limited the utilization of higher dosing. We designed shorter 25-mer PMOs directed to the same eteplirsen-targeted region of exon 51 and compared their efficacies in vitro and in vivo in the mdx52 murine model. Our results showed that skipped-dystrophin induction was comparable between the 30-mer PMO sequence of eteplirsen and one of the shorter PMOs, while the other 25-mer PMOs showed lower exon-skipping efficacies. Shorter PMOs would make higher doses economically feasible, and high dosing would result in better drug uptake into muscle, induce higher levels of dystrophin restoration in DMD muscle, and, ultimately, increase the clinical efficacy.

Keywords: DMD; PMO; dystrophin; eteplirsen; exon skipping; exondys51; mdx; myopathy; phosphorodiamidate morpholino; phosphorothiorate; shorter PMO-SSOs.

Graphical abstract

Figure 1

Representation of Exon 51 Skipping in Dmd Pre-mRNA by PMO-SSOs in H2K-mdx52 Cells (A) Schematic depiction of Dmd pre-mRNA indicates the relative positions of exons (boxes with numbers), flanking introns (blue lines), disruption of the reading frame by the deletion of exon 52 in the H2K-mdx52 cell line, and relative position of the binding site to the PMOs on exon 51 (green line). The exon 52 deletion disrupts the open reading frame. Skipping of exon 51 by Etep and the shorter PMOs restores the downstream reading frame of Dmd mRNA. Arrows on Dmd-mRNAs indicate relative positions of the primers for the nested PCR (black arrows indicate outer primers, and blue arrows indicate inner primers). Nested PCR generates two amplicons, which correspond to unskipped mRNA (422 bp) and skipped mRNA (189 bp). (B) The target sequence within exon 51 of Dmd pre-mRNA and sequences of Etep and shorter PMOs with binding positions. The PMOs are antisense compounds that have reverse-complementary sequences to the target sequence within exon 51.

Figure 2

Nested RT-PCR Analysis of the Transfected H2K mdx52 Cells Showing Exon 51 Skipping Induced by the Etep and the Shorter PMOs Following 24 hr of induction of differentiation, the cells were transfected with the indicated concentrations of the PMOs, and skipping efficacy was analyzed 48 hr post-transfection. Agarose gel electrophoresis shows unskipped and exon-51-skipped transcripts of Dmd (illustrated by boxes with exon numbers). Biological triplicates were performed for each condition. Alpha-actinin (αAct) amplification indicates the proper differentiation of myoblasts to myotubes. First and last lines indicate the DNA ladder (a 100-bp ladder), and NTC is the non-template control. All PMOs exhibited a concentration-dependent increase in exon 51 skipping. (A) Transfection at concentrations ranging from 100 nM to 3,000 nM. (B) Transfection at concentrations ranging from 1 μM to 10 μM.

Figure 3

Exon-51-Skipping Efficacies of Etep and the Shorter PMOs in the H2K mdx52 Cells Agarose gel band intensities of PCR products of Dmd and alpha-actinin were quantified from the corresponding gels (Figure 2A). (A) Percentage of exon 51 skipping induced by each PMO at different concentrations. Skipping percentages were calculated as (normalized relative expression of the skipped transcript/[normalized relative expression of the skipped + normalized relative expression of the unskipped transcripts]) × 100. (B) Dose-response curves of normalized relative expressions of skipped and unskipped Dmd transcripts for the PMOs. The curves of the Dmd transcripts were plotted on the same graphic for each PMO to show and compare the relative expression levels of skipped and unskipped transcripts in response to different concentrations of PMOs. Error bars represent SD.

Figure 4

Validation of Restoration of Dystrophin Production by Shorter PMOs in Dystrophin-Null mdx52 Mice Dystrophin expression in tibialis anterior muscles of mdx52 mice after intramuscular injection of 20 μg of the PMOs. Dystrophin immunoreactivity (Dys, red) was localized to the sarcolemma of myofibers, indicating exon 51 skipping and dystrophin restoration in vivo. Nuclei were stained with DAPI. Scale bars, 200 μm.

Figure 5

Comparison of the Mean Fluorescence of the Dystrophin-Positive Fibers of the PMOs and Saline-Treated mdx52 Mice and Wild-Type Mice Each bar represents the mean fluorescence intensity per fiber. Two mice per group were injected by an indicated dose of the PMOs. Approximately 100 dystrophin-positive fibers were analyzed per mouse. Mean intensities were calculated from the mean intensities of each specimen. Detailed analyses are represented in Figure S4 and Table S3. Wt, wild-type; SC, saline control; E, Etep; D, Etep-downstream; U, Etep-upstream; and M, Etep-middle. *p < 0.05; **p < 0.01. Error bars represent SD.