Original articleIntraperitoneal administration of phosphorothioate antisense oligodeoxynucleotide against splicing enhancer sequence induced exon skipping in dystrophin mRNA expressed in mdx skeletal muscle
Introduction
Duchenne muscular dystrophy (DMD) is a rapid, progressive muscle-wasting disease and is characterized by absence of dystrophin in plasma membrane, while Becker muscular dystrophy (BMD) is a clinically less-severe form of the disease that often has only slight debilitating effects. DMD and BMD are allelic diseases caused by mutations in the dystrophin gene. The clinical progression of DMD or BMD patients can be predicted from whether the deletion disrupts (out-of-frame) or maintains (in-frame) the translational reading frame of the mRNA (frame shift theory) [1].
Much progress has been made in the study of gene therapy for DMD. Most DMD gene therapy have been based on replacement of the dystrophin gene, but has been hampered by the large size of the dystrophin gene and the absence of an efficient systemic delivery system for the transfected gene. Therefore, an alternative strategy for DMD treatment might be to retard the progression of the clinical symptoms, i.e. to convert DMD into BMD phenotype. Theoretically this therapy could be achieved by modification of out-of-frame deletion causing DMD into in-frame deletion characteristics of BMD by modifying the dystrophin mRNA.
Artificial induction of exon skipping at the time of splicing is one choice to correct out-of-frame into in-frame. In a previous study, we demonstrated that disruption of the splicing enhancer sequence (SES) of exon 19 by a complementary antisense oligodeoxynucleotide resulted in complete skipping of exon 19 in Epstein–Barr virus transformed lymphoblastoid cells established from normal human cells [2]. Furthermore, the antisense oligodeoxynucleotide induced exon 19 skipping successfully in dystrophin mRNA from DMD myocytes with exon 20 deletion. Skipping of exon 19 in mRNA with exon 20 deletion produced an in-frame dystrophin mRNA with continuous exon 19 and 20 deletion resulting in production of dystrophin protein from DMD myocytes [3]. Recently, antisense oligonucleotide against polypurine sequence of exon 46 was shown to induce skipping of exon 46 from mature dystrophin mRNA [4]. Furthermore, antisense oligonucleotides against intra-exon sequences of other exons of the dystrophin gene have also been reported to induce exon skipping in cultured muscle cells [5]. These results raised the possibility that the antisense oligodeoxynucleotide could be used clinically to correct the translational reading frame of DMD dystrophin mRNA at the splicing step. However, there is no established way to transfer oligodeoxynucleotide into diseased muscle cells. This has hampered the application of oligodeoxynucleotide to clinical use.
Mdx mouse is a mouse model of DMD and no dystrophin is stained on the plasma membrane of the skeletal muscle due to a nonsense mutation in the exon 23 of the dystrophin gene [6]. The functional defects of plasma membrane have been shown by leakage of cytosolic enzymes such as creatine kinase into the blood flow. On the other hand, incorporation of materials in the blood stream was demonstrated by Evans blue staining of skeletal muscle cells after an intravenous injection of the dye [7]. These findings indicated the passage of low molecular weight material through the damaged plasma membrane of mdx mouse.
Here, we reported the first evidence that phosphorothioate antisense oligodeoxynucleotide without any carrier is incorporated into the nucleus of skeletal muscle of mdx mouse. Furthermore, the expected effect of transfection of the antisense oligodeoxynucleotide was demonstrated using molecular biological techniques.
Section snippets
Oligodeoxynucleotide
The 31-mer phosphorothioate oligodeoxynucleotide (5′-GCCTGAGCTGATCTGCTGGCATCTTGCAGTT-3′) complementary to the first 31 nucleotides of the deleted region in exon 19 of dystrophin Kobe [8] was synthesized and dissolved in sterile phosphate-buffer saline at 10 mg/ml. It has been shown that the oligodeoxynucleotide can inhibit in vitro splicing of intron 18 transcribed from the minigene [9] and induce exon 19 skipping in EBV-transformed normal human lymphoblastoid cells [2] and cultured DMD myocytes
Lacalization of oligodeoxynucleotide in muscle after intraperitoneal administration
In order to trace the movement of oligodeoxynucleotide, the FITC labeled phosphorothioate antisense oligodeoxynucleotide against SES in exon 19 of the dystrophin gene was administered intraperitoneally to three groups of mdx mice at a dose of 20 mg/kg BW. The mice were sacrificed on the second day after administration, and remaining groups were sacrificed on the fourth and 14th day after administration. Sections of femoral skeletal muscle were frozen for microscopic analysis. Under Zeiss
Discussion
Transfection of phosphorothioate antisense oligodeoxynucleotide was studied in this report using DMD mouse model, which lacks dystrophin completely. Remarkably, oligodeoxynucleotide was efficiently transfected into the nuclei of skeletal muscle without any carrier. This was clearly demonstrated by the appearance of an FITC signal in nuclei. Furthermore, the expected effect of transfection of the antisense oligodeoxynucleotide was demonstrated by showing exon 19 skipping of the dystrophin
Acknowledgements
This work was supposed by grants from the Ministry of Education, Science, Spots and Culture of Japan and the National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare of Japan.
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2008, Neuromuscular DisordersCitation Excerpt :Although this correction is only temporary, it induces improved function of the patient cells and mouse muscle. More recently, the repeated systemic (intravenous) administration of AONs was shown to be capable of restoring a sustained dystrophin expression in the mouse model of DMD, and this was followed by a significant functional improvement of the mouse muscle function [3–6]. If safe and equally effective in people, the repeated systemic administration of AONs could therefore be an effective tool to slow down the disease progression in DMD boys.
Non-viral gene therapy for Duchenne muscular dystrophy: Progress and challenges
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