Optimization of peptide nucleic acid antisense oligonucleotides for local and systemic dystrophin splice correction in the mdx mouse

Abstract

Antisense oligonucleotides (AOs) have the capacity to alter the processing of pre-mRNA transcripts in order to correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease that arises from mutations in the DMD gene leading to an absence of dystrophin protein. AOs have been shown to restore the expression of functional dystrophin via splice correction by intramuscular and systemic delivery in animal models of DMD and in DMD patients via intramuscular administration. Major challenges in developing this splice correction therapy are to optimize AO chemistry and to develop more effective systemic AO delivery. Peptide nucleic acid (PNA) AOs are an alternative AO chemistry with favorable in vivo biochemical properties and splice correcting abilities. Here, we show long-term splice correction of the DMD gene in mdx mice following intramuscular PNA delivery and effective splice correction in aged mdx mice. Further, we report detailed optimization of systemic PNA delivery dose regimens and PNA AO lengths to yield splice correction, with 25-mer PNA AOs providing the greatest splice correcting efficacy, restoring dystrophin protein in multiple peripheral muscle groups. PNA AOs therefore provide an attractive candidate AO chemistry for DMD exon skipping therapy.

Figure 1

Restoration of dystrophin expression in aged mdx mice. Restoration of dystrophin expression in aged mdx mice following single 5-µg intramuscular injections of PNA AOs in 12-month-old mdx mice. (a) Immunofluorescent staining for dystrophin in mdx TA muscles following 5 µg injection of PNA AO and peptide-PNA conjugates. Nuclei were counterstained with 4′,6-diamidino-2 phenylindole. (b) Quantitative evaluation of total dystrophin-positive fibers in treated TA muscles 2 weeks after single intramuscular injection of 5 µg PNA AO and MSP- and AAV6-PNA conjugates. Neutral PNA AO showed significant increase of dystrophin-positive fibers compared with untreated age-match control and peptide-PNA conjugates (*P < 0.05). (c) RT-PCR for detecting exon skipping efficiency at the RNA level, which is shown by shorter exon-skipped bands (indicated by the numbered δexon23^– for exon 23 skipping). AAV, adeno-associated virus; AO, antisense oligonucleotide; MSP, muscle-specific peptide; PNA, peptide nucleic acid; RT-PCR, reverse transcription-PCR; TA, tibialis anterior.

Figure 2

Long-term correction of dystrophin expression following intramuscular PNA administration in mdx mice. (a) Immunohistochemistry for dystrophin induction in TA muscles of 2-month-old mdx mice 2, 4, 8, 16, and 20 weeks after one single intramuscular injection of 5 µg PNA AO (bar = 200 µm). (b) Quantitative evaluation of total dystrophin-positive fibers in TA muscles at different time points after a single intramuscular injection of PNA AO. All groups showed significant improvement in comparison with age-matched untreated mdx mice (*P < 0.05). (c) RT-PCR to detect exon skipping efficiency at the RNA level demonstrated up to 50% exon 23 skipping in the TA muscle treated with PNA AO at 4 weeks after injection. This is shown by shorter exon-skipped bands (indicated by δexon23^– for exon 23 skipping). (d) Western blot analysis. Western blot for treated TA muscles at different time points after single intramuscular injection of PNA AOs. Total protein was extracted from TA muscles of 2-month-old mdx mice at 2, 4, 8, 16, and 20 weeks after a single intramuscular injection with 5 µg PNA AOs. Fifty microgram of total protein from untreated mdx mice TA muscles and treated muscle samples was loaded. 2.5- and 0.5-µg total protein (5 and 1%, respectively) from C57BL6 TA muscles were loaded as normal controls. No visible difference in the size of dystrophins between muscle treated with PNA and muscle from the normal C57BL6 mouse. α-Actinin was used as a loading control. AO, antisense oligonucleotide; PNA, peptide nucleic acid; RT-PCR, reverse transcription-PCR; TA, tibialis anterior.

Figure 3

Systemic delivery of PNA AOs for dystrophin splice correction in mdx mice. (a) Immunohistochemistry for dystrophin induction in body-wide muscles of 2-month-old mdx mice 2 weeks after one single intravenous injection of PNA20 AOs at 100 mg/kg dose (bar = 200 µm). (b) Quantitative evaluation of total dystrophin-positive fibers in all tested muscles from TA, quadriceps, gastrocnemius, biceps, diaphragm, heart, and abdominal wall 2 weeks after single intravenous injection of different doses of PNA AOs. The data indicated significant difference between the treatments with PNA20 AOs at different doses compared with untreated age-matched control mdx mice (*P < 0.05). AO, antisense oligonucleotide; PNA, peptide nucleic acid; TA, tibialis anterior.

Figure 4

Effects of PNA length on splice correction activity in mdx mice. We evaluated the effect of AO length on PNA splice correcting activity in mdx mice by studying the activities of seven PNA compounds of 15, 16, 17, 18-1, 18-2, 20, and 25 nucleotides in length, respectively (see Table 1). (a) Immunohistochemistry for dystrophin induction in TA muscles of 2-month-old mdx mice 2 weeks after one single intramuscular injection of 5 µg different lengths of PNA AO (bar = 200 µm). (b) Quantitative evaluation of total dystrophin-positive fibers in TA muscles 2 weeks after a single intramuscular injection of various PNA AOs. TA muscles treated with PNA25 showed significant improvement in comparison with age-matched untreated mdx mice and other PNA AOs (*P < 0.05). (c) RT-PCR to detect exon skipping efficiency at the RNA level demonstrated up to 50% exon 23 skipping in the TA muscle treated with PNA25 AO at 2 weeks after injection. This is shown by shorter exon-skipped bands (indicated by δexon23^– for exon 23 skipping). (d) Western blot analysis. Western blot for treated TA muscles after single intramuscular injection of various PNA AOs. Total protein was extracted from TA muscles of 2-month-old mdx mice 2 weeks after a single intramuscular injection with 5 µg different PNA AOs. Fifty microgram total protein from untreated mdx mice TA muscles and treated muscle samples was loaded. 2.5- and 0.5-µg total protein (5 and 1%, respectively) from C57BL6 TA muscles were loaded as normal controls. No visible difference in the size of dystrophins between muscle treated with PNA and muscle from the normal C57BL6 mouse. α-Actinin was used as a loading control. AO, antisense oligonucleotide; PNA, peptide nucleic acid; RT-PCR, reverse transcription-PCR; TA, tibialis anterior.

Figure 5

Systemic restoration of dystrophin expression by 25-mer PNA AO compounds. We evaluated the systemic splice correcting ability of PNA25 compounds following their improved activity in an intramuscular screen. (a) Quantitative evaluation of total dystrophin-positive fibers in all tested muscles from TA, quadriceps, gastrocnemius, biceps, diaphragm, heart, and abdominal wall 2 weeks after single intravenous injection of PNA AOs. The data indicated that there was significant difference between the treatment with PNA25 and PNA20 AOs in some muscle groups (*P < 0.05) (b) RT-PCR to detect exon skipping efficiency at the RNA level demonstrated up to 10% exon 23 skipping in the quadriceps muscle treated with PNA25 AO. This is shown by shorter exon-skipped bands (indicated by δexon23^– for exon 23 skipping). AO, antisense oligonucleotide; PNA, peptide nucleic acid; RT-PCR, reverse transcription-PCR; TA, tibialis anterior.