Splicing reporter minigenes are used in cell-based in vitro splicing studies. Exon skippable antisense oligonucleotide (ASO) has been identified using minigene splicing assays, but these assays include a time- and cost-consuming step of reverse transcription PCR amplification. To make in vitro splicing assay easier, a ready-made minigene (FMv2) amenable to quantitative splicing analysis by fluorescence microscopy was constructed. FMv2 was designed to encode two fluorescence proteins namely, mCherry, a transfection marker and split eGFP, a marker of splicing reaction. The split eGFP was intervened by an artificial intron containing a multicloning site sequence. Expectedly, FMv2 transfected HeLa cells produced not only red mCherry but also green eGFP signals. Transfection of FMv2CD44v8, a modified clone of FMv2 carrying an insertion of CD44 exon v8 in the multicloning site, that was applied to screen exon v8 skippable ASO, produced only red signals. Among seven different ASOs tested against exon v8, ASO#14 produced the highest index of green signal positive cells. Hence, ASO#14 was the most efficient exon v8 skippable ASO. Notably, the well containing ASO#14 was clearly identified among the 96 wells containing randomly added ASOs, enabling high throughput screening. A ready-made FMv2 is expected to contribute to identify exon skippable ASOs.

Antisense oligonucleotides (ASOs) are agents that modulate gene function. ASO-mediated out-of-frame exon skipping has been employed to suppress gene function. Myostatin, encoded by the MSTN gene, is a potent negative regulator of skeletal muscle growth. ASOs that induce skipping of out-of-frame exon 2 of the MSTN gene have been studied for their use in increasing muscle mass. However, no ASOs are currently available for clinical use. We hypothesized that ASOs against the splicing enhancer sequence within exon 1 of the MSTN gene would inhibit maturation of pre-mRNA, thereby suppressing gene function. To explore this hypothesis, ASOs against sequences of exon 1 of the MSTN gene were screened for their ability to reduce mature MSTN mRNA levels. One screened ASO, named KMM001, decreased MSTN mRNA levels in a dose-dependent manner and reciprocally increased MSTN pre-mRNA levels. Accordingly, KMM001 decreased myostatin protein levels. KMM001 inhibited SMAD-mediated myostatin signaling in rhabdomyosarcoma cells. Remarkably, it did not decrease GDF11 mRNA levels, indicating myostatin-specific inhibition. As expected, KMM001 enhanced the proliferation of human myoblasts. We conclude that KMM001 is a novel myostatin inhibitor that inhibits pre-mRNA maturation. KMM001 has great promise for clinical applications and should be examined for its ability to treat various muscle-wasting conditions.

The DMD gene is one of the largest human genes, being composed of 79 exons, and encodes dystrophin Dp427m which is deficient in Duchenne muscular dystrophy (DMD). In some DMD patient, however, small size dystrophin reacting with antibody to N-terminal but not to C-terminal has been identified. The mechanism to produce N-terminal small size dystrophin remains unknown. Intronic polyadenylation is a mechanism that produces a transcript with a new 3′ terminal exon and a C-terminal truncated protein. In this study, intronic alternative polyadenylation was disclosed to occur in the middle of the DMD gene and produce the half-size N-terminal dystrophin Dp427m, Dpm234. The 3′-rapid amplification of cDNA ends revealed 421 bp sequence in the downstream of DMD exon 41 in U-251 glioblastoma cells. The cloned sequence composing of the 5′ end sequence of intron 41 was decided as the terminal exon, since it encoded poly (A) signal followed by poly (A) stretch. Subsequently, a fragment from DMD exon M1 to intron 41 was obtained by PCR amplification. This product was named Dpm234 after its molecular weight. However, Dpm234 was not PCR amplified in human skeletal and cardiac muscles. Remarkably, Dpm234 was PCR amplified in iPS-derived cardiomyocytes. Accordingly, Western blotting of cardiomyocyte proteins showed a band of 234 kDa reacting with dystrophin antibody to N-terminal, but not C-terminal. Clinically, DMD patients with mutations in the Dpm234 coding region were found to have a significantly higher likelihood of two ECG abnormal findings. Intronic alternative splicing was first revealed in Dp427m to produce small size dystrophin.