Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice
Authors: Natee Jearawiriyapaisarn, Hong M Moulton, Brian Buckley, Jennifer Roberts, Peter Sazani, Suthat Fucharoen, Patrick L Iversen, Ryszard Kole
Journal: Molecular Therapy (2008)

Cell-penetrating peptides (CPPs), containing arginine (R), 6-aminohexanoic acid (X), and/or β-alanine (B) conjugated to phosphorodiamidate morpholino oligomers (PMOs), enhance their delivery in cell culture. In this study, the potency, functional biodistribution, and toxicity of these conjugates were evaluated in vivo, in EGFP-654 transgenic mice that ubiquitously express the aberrantly spliced EGFP-654 pre-mRNA reporter. Correct splicing and enhanced green fluorescence protein (EGFP) upregulation serve as a positive readout for peptide-PMO (PPMO) entry into cells and access to EGFP-654 pre-mRNA in the nucleus. Intraperitoneal injections of a series of PPMOs, A-N (12 mg/kg), administered once a day for four successive days resulted in splicing correction in numerous tissues. PPMO-B was highly potent in the heart, diaphragm, and quadriceps, which are key muscles in the treatment of Duchenne muscular dystrophy. We therefore investigated PPMO M23D-B, designed to force skipping of stop-codon containing dystrophin exon 23, in an mdx mouse model of the disease. Systemic delivery of M23D-B yielded persistent exon 23 skipping, yielding high and sustained dystrophin protein expression in body-wide muscles, including cardiac muscle, without detectable toxicity. The rescued dystrophin reduced serum creatinine kinase to near-wild-type levels, indicating improvement in muscle integrity. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart of treated animals. Cell-penetrating peptides (CPPs), containing arginine (R), 6-aminohexanoic acid (X), and/or β-alanine (B) conjugated to phosphorodiamidate morpholino oligomers (PMOs), enhance their delivery in cell culture. In this study, the potency, functional biodistribution, and toxicity of these conjugates were evaluated in vivo, in EGFP-654 transgenic mice that ubiquitously express the aberrantly spliced EGFP-654 pre-mRNA reporter. Correct splicing and enhanced green fluorescence protein (EGFP) upregulation serve as a positive readout for peptide-PMO (PPMO) entry into cells and access to EGFP-654 pre-mRNA in the nucleus. Intraperitoneal injections of a series of PPMOs, A-N (12 mg/kg), administered once a day for four successive days resulted in splicing correction in numerous tissues. PPMO-B was highly potent in the heart, diaphragm, and quadriceps, which are key muscles in the treatment of Duchenne muscular dystrophy. We therefore investigated PPMO M23D-B, designed to force skipping of stop-codon containing dystrophin exon 23, in an mdx mouse model of the disease. Systemic delivery of M23D-B yielded persistent exon 23 skipping, yielding high and sustained dystrophin protein expression in body-wide muscles, including cardiac muscle, without detectable toxicity. The rescued dystrophin reduced serum creatinine kinase to near-wild-type levels, indicating improvement in muscle integrity. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart of treated animals. IntroductionIn order to enhance the efficacy of antisense oligonucleotides as therapeutic agents, numerous chemical modifications have been introduced to their bases, backbones, and internucleotide linkages.1Kurreck J Antisense technologies. Improvement through novel chemical modifications.Eur J Biochem. 2003; 270: 1628-1644Crossref PubMed Scopus (949) Google Scholar One such modification, phosphorodiamidate morpholino oligomers (PMOs),2Summerton J Morpholino antisense oligomers: the case for an RNase H-independent structural type.Biochim Biophys Acta. 1999; 1489: 141-158Crossref PubMed Scopus (554) Google Scholar,3Arora V Devi GR Iversen PL Neutrally charged phosphorodiamidate morpholino antisense oligomers: uptake, efficacy and pharmacokinetics.Curr Pharm Biotechnol. 2004; 5: 431-439Crossref PubMed Scopus (65) Google Scholar bind their RNA targets with high affinity and specificity, and are highly resistant to nucleolytic degradation; however, their systemic distribution and functionality were found to be modest in mouse model systems.4Sazani P Gemignani F Kang SH Maier MA Manoharan M Persmark M et al.Systemically delivered antisense oligomers upregulate gene expression in mouse tissues.Nat Biotechnol. 2002; 20: 1228-1233Crossref PubMed Scopus (269) Google Scholar Subsequently, conjugates of PMOs with arginine-rich cell-penetrating peptides (CPPs), termed peptide-PMOs (PPMOs), were found to possess enhanced potency in vitro.5Abes R Arzumanov AA Moulton HM Abes S Ivanova GD Iversen PL et al.Cell-penetrating-peptide-based delivery of oligonucleotides: an overview.Biochem Soc Trans. 2007; 35 (Pt 4): 775-779Crossref Scopus (102) Google Scholar,6Lebleu B Moulton HM Abes R Ivanova GD Abes S Stein DA et al.Cell penetrating peptide conjugates of steric block oligonucleotides.Adv Drug Deliv Rev. 2007; 60: 517-529Crossref PubMed Scopus (156) Google Scholar,7Abes R Arzumanov A Moulton H Abes S Ivanova G Gait MJ et al.Arginine-rich cell penetrating peptides: design, structure-activity, and applications to alter pre-mRNA splicing by steric-block oligonucleotides.J Pept Sci. 2008; 14: 455-460Crossref PubMed Scopus (58) Google Scholar,8Wu RP Youngblood DS Hassinger JN Lovejoy CE Nelson MH Iversen PL et al.Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity.Nucleic Acids Res. 2007; 35: 5182-5191Crossref PubMed Scopus (94) Google Scholar In particular, serum and intracellular stability of peptides was increased, endosomal trapping was reduced, and nuclear uptake was enhanced by the introduction of non-α amino acids, namely, 6-aminohexanoic acid (X) and β-alanine (B), into the oligoarginine (R8) peptides.9Moulton HM Nelson MH Hatlevig SA Reddy MT Iversen PL Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides.Bioconjug Chem. 2004; 15: 290-299Crossref PubMed Scopus (170) Google Scholar,10Amantana A Moulton HM Cate ML Reddy MT Whitehead T Hassinger JN et al.Pharmacokinetics, biodistribution, stability and toxicity of a cell-penetrating peptide-morpholino oligomer conjugate.Bioconjug Chem. 2007; 18: 1325-1331Crossref PubMed Scopus (143) Google Scholar,11Youngblood DS Hatlevig SA Hassinger JN Iversen PL Moulton HM Stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells.Bioconjug Chem. 2007; 18: 50-60Crossref PubMed Scopus (140) Google Scholar,12Abes S Moulton HM Clair P Prevot P Youngblood DS Wu RP et al.Vectorization of morpholino oligomers by the (R-Ahx-R)4 peptide allows efficient splicing correction in the absence of endosomolytic agents.J Control Release. 2006; 116: 304-313Crossref PubMed Scopus (164) Google ScholarIn this study, a series of PPMOs carrying CPPs consisting of R8 residues and a variable number of X and/or B residues (termed A through N, Table 1) was designed. The series was first evaluated in EGFP-654 transgenic mice, which ubiquitously express an enhanced green fluorescence protein (EGFP) pre-mRNA interrupted by an aberrantly spliced human β-globin IVS2-654 intron. The EGFP upregulation serves as a positive readout for the ability of PPMOs to enter cells and correct aberrant splicing of the EGFP-654 pre-mRNA in the nucleus4Sazani P Gemignani F Kang SH Maier MA Manoharan M Persmark M et al.Systemically delivered antisense oligomers upregulate gene expression in mouse tissues.Nat Biotechnol. 2002; 20: 1228-1233Crossref PubMed Scopus (269) Google Scholar (Figure 1a). After delivery through intraperitoneal (IP) injection, all PPMOs exhibited a broad tissue distribution and corrected the aberrant splicing in EGFP-654 mice to varying degrees.Table 1Nomenclature and sequences of CPPsNameSequenceAbbreviationLengthARRRRRRRRXB(R)8xb10BRXRRBRRXRRBRXB(rxrrbr)2xb14CRXRRXRRXRRBRXB(RXR)3RBRXB14DRBRBRBRBRBRXRBRXB(rb)5rxrbrxb17ERBRBRBRXRBRBRBRXX(RB)3RX(RB)3RXX17FXRBRBRBRXRBRBRBRXX(RB)3RX(RB)3RX17GRBRXRBRXRBRXRBRXB(rbrx)4b17HRBRBRBRBRXRXRXRXB(RB)4(RX)4B17IRXRBRBRXRBRBRBRXXRX(RB)2RX(RB)3RXX17JrXrrXrrXrrXrXB(rXr)4XB14KRXRRXRRXRRXRXB(RXR)4XB14LRBRBRBRBRBRBRBRBB(RB)8B17MRBRBRBRBRBRBRBRXB(rb)7rxb17NRBRBRBRBRBRBRBRBB(RB)8B17Abbreviation: CPP, cell-penetrating peptide.B, β-alanine; r, D-arginine; R, L-arginine; X, 6-aminohexanoic acid. Open table in a new tab One of the conjugates, PPMO-B, was very effective in the heart, diaphragm, and quadriceps, the target muscles for treatment of Duchenne muscular dystrophy (DMD), a severe progressive muscle wasting disorder.13Lovering RM Porter NC Bloch RJ The muscular dystrophies: from genes to therapies.Phys Ther. 2005; 85: 1372-1388PubMed Google Scholar This work focused on the use of PPMO-B in the treatment of DMD in an mdx mouse model of the disease. We found that systemic delivery of a PPMO-B targeted to exon 23 of the dystrophin gene (M23D-B) induced high and sustained dystrophin protein expression in body-wide muscles in treated mdx mice. This led to improved cardiac histology and a dramatic drop in serum creatinine kinase (CK), a hallmark of improvement in muscle integrity.ResultsFunctional biodistribution of PPMO-654 conjugates in EGFP-654 miceIn order to evaluate the in vivo potency and functional biodistribution of PPMOs, EGFP-654 mice were injected IP once a day for 4 days at 12 mg/kg/day with PPMO-654 conjugates (A through N, Table 1), targeted to the aberrant 5'-splice site in EGFP-654 pre-mRNA (Figure 1a). One day after the last injection, the mice were killed, and total RNA was isolated from tissues and analyzed using reverse transcriptase-PCR (RT-PCR). The results show that all the PPMO-654 conjugates exhibited broad tissue-wide distribution, with significant splicing correction in most tissues (Figure 1b) (but not in brain and bone marrow; Supplementary Figure S1a), which was substantially higher than that achieved with higher doses of unconjugated PMOs.4Sazani P Gemignani F Kang SH Maier MA Manoharan M Persmark M et al.Systemically delivered antisense oligomers upregulate gene expression in mouse tissues.Nat Biotechnol. 2002; 20: 1228-1233Crossref PubMed Scopus (269) Google ScholarThe number and position of 6-aminohexanoic acid (X) and/or β-alanine (B) residues inserted in the oligoarginine (R8) in the CPPs significantly affected the potency and functional biodistribution of the PPMOs. PPMO-A, containing 8 arginines, 1 X residue and 1 B residue was very effective in modifying EGFP-654 pre-mRNA splicing in liver (∼63%), but was much less effective in several other tissues including the quadriceps and the heart (Figure 1b). In contrast, PPMO-B was the most effective in the quadriceps (100%) and the heart (59%), but its efficacy in the liver was similar to that of PPMO-A. PPMO-J was highly effective in the kidney (100%) and quadriceps (100%) but not in the heart (21%). Surprisingly, all the PPMOs corrected the EGFP-654 splicing in diaphragm 100%. Overall, the differences in the functional biodistribution of PMOs conjugated with a family of closely related peptides are striking, and more structure-activity studies are needed to elucidate the causes and mechanisms of these differences.We noticed that PPMO-B is the most potent in all muscle tissues, especially the heart. PPMO-B did not produce toxicity in mice at the doses tested (Figure 1c and Supplementary Figure S1b), and the data in Figure 2a and b show a clear dose–response in the heart and quadriceps, while in the diaphragm, even at the low dose of 3 mg/kg, 100% of correctly spliced EGFP mRNA was produced. Therefore, PPMO-B represents a promising candidate for the treatment of DMD. The strategy developed in our laboratory14Sierakowska H Sambade MJ Agrawal S Kole R Repair of thalassemic human β-globin mRNA in mammalian cells by antisense oligonucleotides.Proc Natl Acad Sci USA. 1996; 93: 12840-12844Crossref PubMed Scopus (204) Google Scholar,15Wilton SD Lloyd F Carville K Fletcher S Honeyman K Agrawal S et al.Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides.Neuromuscul Disord. 1999; 9: 330-338Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar is to induce in-frame skipping of an offending exon which causes a premature termination codon, restoring production of functional dystrophin protein (Supplementary Figure S2).Figure 2Effective and persistent splicing correction of PPMO-B conjugate in the heart, diaphragm (diap), and quadriceps (quad) of EGFP-654 mice. (a–b) Dose-dependent splicing correction. EGFP-654 mice were treated with PPMO-B conjugate at the indicated doses by intravenous (IV) injection once every day for 4 days. (a) Reverse transcriptase-PCR (RT-PCR) of total RNA and (b) fluorescent images of tissue sections were analyzed 1 day after the last injection. Samples A and B are from different mice. (Scale bar = 100 μm). (c–d) Time-dependent splicing correction. The EGFP-654 mice were injected IV with PPMO-B conjugate at 12 mg/kg/day for 4 days. (c) Total RNA samples from the heart, diap, and quad were analyzed using RT-PCR at the indicated time points. (d) Upregulation of enhanced green fluorescence protein (EGFP) expression was monitored using a fluorescent live imaging system. WT is a mouse that constitutively expresses EGFP. The EGFP-654 mice treated with PPMO-B, the control mice treated with PPMO-B at 12 mg/kg, and untreated mice are shown as B, scr-B, and un, respectively. PPMO, peptide-phosphorodiamidate morpholino oligomer.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In order to determine the persistence of PPMO-B activity, EGFP-654 mice were injected intravenously (IV) at 12 mg/kg/day for 4 days, and killed up to 12 weeks after the last injection (Figure 2c); IV administration of PPMO-B was more efficient than IP and subcutaneous routes, especially in the heart (Supplementary Figure S3). The splicing correction in the heart reached 90% at 1 day after the last injection and then gradually decreased, but was still detectable (∼10%) at 6 weeks. In the diaphragm, 100% correctly spliced EGFP mRNA was detectable up to 2–3 weeks after the last injection, decreasing to 10–40% at 12 weeks. Impressively, PPMO-B exhibited 100% splicing correction in the quadriceps up to 12 weeks after the last injection.The expression of EGFP was also followed-up in live animals using the Xenogen IVIS 100 imaging system (Figure 2d). The EGFP signal was detectable in the abdominal fascia and quadriceps from 1 week after the last injection, and increased for up to 5 weeks without any decrease in fluorescence intensity at week 8. A PPMO targeted to mouse dystrophin with three mismatches (PPMO mis-23D), used as a negative control, did not upregulate EGFP.Skipping of dystrophin exon 23 in mdx mice by PPMO M23D-BThe phenotype of the mdx mouse is caused by a premature termination codon in exon 23 of the dystrophin gene (Supplementary Figure S2), which prevents translation of dystrophin protein. Oligonucleotide-induced skipping of exon 23 during splicing of dystrophin pre-mRNA restores the reading frame in the spliced mRNA, resulting in the production of partially functional truncated dystrophin protein in the skeletal muscles of mdx mice.16Mann CJ Honeyman K Cheng AJ Ly T Lloyd F Fletcher S et al.Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse.Proc Natl Acad Sci USA. 2001; 98: 42-47Crossref PubMed Scopus (348) Google Scholar,17Lu QL Mann CJ Lou F Bou-Gharios G Morris GE Xue SA et al.Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse.Nat Med. 2003; 9: 1009-1014Crossref PubMed Scopus (330) Google Scholar,18Lu QL Rabinowitz A Chen YC Yokota T Yin H Alter J et al.Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles.Proc Natl Acad Sci USA. 2005; 102: 198-203Crossref PubMed Scopus (349) Google Scholar Recently, the systemically delivered unconjugated morpholino oligonucleotide, PMO M23D, targeted to a donor splice site of mouse dystrophin intron 23, was found to be effective in skeletal muscles but, importantly, not in the heart.19Alter J Lou F Rabinowitz A Yin H Rosenfeld J Wilton SD et al.Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology.Nat Med. 2006; 12: 175-177Crossref PubMed Scopus (431) Google Scholar,20Fletcher S Honeyman K Fall AM Harding PL Johnsen RD Wilton SD Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide.J Gene Med. 2006; 8: 207-216Crossref PubMed Scopus (160) Google Scholar Given that, as described earlier in the text, PPMO-B was highly efficient and persistent in splicing correction in the heart, diaphragm, and quadriceps in EGFP-654 mice, we synthesized the PPMO M23D-B conjugate for treating mdx mice.The mdx mice were injected IV once every day for 4 days with M23D-B at 12 mg/kg/day (Figure 3). Muscle tissues were collected and analyzed at the indicated time points for exon skipping, by nested RT-PCR of dystrophin mRNA. In the heart, the exon 23–skipped dystrophin mRNA reached 70% at 1 day after the last injection and decreased to 50 and 20% at 2 and 7 weeks, respectively; at 9 weeks after the last injection, exon 23 skipping was still detectable. In the diaphragm and quadriceps, M23D-B induced almost complete exon 23 skipping, and this remained essentially unchanged up to 9 weeks after the last injection. Similar activity was detected in all the other skeletal muscles (Supplementary Figure S4). No exon 23–skipped transcript was observed in saline or mismatched oligo-treated mdx mice or in wild-type C57BL6 mice (data not shown).Figure 3Persistence of M23D-B-induced exon 23 skipping of dystrophin transcript in the heart, diaphragm (diap), and quadriceps (quad) of mdx mice after intravenous injection at 12 mg/kg/day for 4 days. RNA samples were analyzed using nested reverse transcriptase-PCR (RT-PCR) at the indicated time points. The nested RT-PCR product from the full-length dystrophin transcript is 445 base pairs (bp), indicated by boxes numbered 21–24. The product from exon 23–skipped mRNA is 232 bp, as indicated by boxes numbered 21, 22, and 24.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Analysis of total protein from M23D-B-treated mice by in-gel immunostaining with anti-dystrophin antibody (Figure 4a) showed that, in the heart, the dystrophin levels reached ∼30% of wild-type at 2–3 weeks and remained at ∼15% up to 7 weeks after the last injection. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart. In the diaphragm and quadriceps, significant dystrophin production, 40–50% of wild type, was achieved and maintained for up to 17 weeks after the last injection (Figure 4a). The multiple dystrophin bands detected in samples from wild-type and treated mdx mice were naturally occurring variants or synthetic intermediates of dystrophin.21Nicholson LV Davison K Falkous G Harwood C O'Donnell E Slater CR et al.Dystrophin in skeletal muscle. I. Western blot analysis using a monoclonal antibody.J Neurol Sci. 1989; 94: 125-136Abstract Full Text PDF PubMed Scopus (158) Google Scholar These results clearly confirm that the persistent skipping of exon 23 induced by M23D-B resulted in robust translation of dystrophin protein, which persisted for up to 17 weeks after the last injection, and localized correctly at the periphery of muscle fibers (Figure 4b). As expected, no detectable dystrophin protein was observed in untreated mdx mice.Figure 4Restoration of dystrophin (Dys) expression in the heart, diaphragm (diap), and quadriceps (quad) of M23D-B-treated mice after intravenous injections at 12 mg/kg/day for 4 days. (a) In-gel western blot detection of total protein extracted from injected mdx muscles at the indicated time points. Dys protein was detected by mouse monoclonal antibody against C-terminus of Dys, followed by IRDye 680 goat anti-mouse immunoglobulin G (IgG) antibody. The three different Dys bands represent naturally occurring variants or synthetic intermediates of Dys. (b) Immunofluorescence detection of Dys on frozen muscle sections from the heart, diap, and quad of treated mdx mice. Sections were immunostained for Dys with NCL-DYS2 and detected using biotinylated anti-mouse IgG and fluorescein avidin. Sections from the heart, diap, and quad were analyzed 3, 2, and 2 weeks after the last injection, respectively. Muscle sections from C57BL and untreated mdx mice were included as positive and negative controls, respectively. Scale bar, 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The discrepancy between the level of exon skipping in mRNA (100%) and the level of induced dystrophin protein (40–50%) relative to wild type suggests that RT-PCR overestimates the extent of exon-skipped mRNA. This is not surprising, considering that nested PCRs were required. Note, however, that we used a total of 30 cycles in the PCR, which compares favorably with published 55 cycle nested PCR protocols.18Lu QL Rabinowitz A Chen YC Yokota T Yin H Alter J et al.Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles.Proc Natl Acad Sci USA. 2005; 102: 198-203Crossref PubMed Scopus (349) Google Scholar It is also possible that the full-length dystrophin mRNA carrying premature termination codon in exon 23 may be degraded by nonsense-mediated decay, resulting in overestimation of corrected dystrophin mRNA. Nevertheless, the important observation is that therapeutically relevant levels22Wells DJ Wells KE Asante EA Turner G Sunada Y Campbell KP et al.Expression of human full-length and minidystrophin in transgenic mdx mice: implications for gene therapy of Duchenne muscular dystrophy.Hum Mol Genet. 1995; 4: 1245-1250Crossref PubMed Scopus (147) Google Scholar,23Phelps SF Hauser MA Cole NM Rafael JA Hinkle RT Faulkner JA et al.Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice.Hum Mol Genet. 1995; 4: 1251-1258Crossref PubMed Scopus (264) Google Scholar of dystrophin were produced in all key muscle types: skeletal muscle, diaphragm, and heart.The sera collected during the experiment were tested for CK, a marker for heart and skeletal muscle membrane instability.24Glesby MJ Rosenmann E Nylen EG Wrogemann K Serum CK, calcium, magnesium, and oxidative phosphorylation in mdx mouse muscular dystrophy.Muscle Nerve. 1988; 11: 852-856Crossref PubMed Scopus (73) Google Scholar,25McArdle A Edwards RH Jackson MJ How does dystrophin deficiency lead to muscle degeneration?—evidence from the mdx mouse.Neuromuscul Disord. 1995; 5: 445-456Abstract Full Text PDF PubMed Scopus (84) Google Scholar Figure 5a demonstrates a dramatic reduction in the level of serum CK from ∼30,000 to <2,000 U/l 1 day after the last injection. Commensurate with additional increases in dystrophin in subsequent weeks (Figure 4a), serum CK levels were further reduced to ∼500 U/l. This level was maintained for 11 weeks after the last injection, and increased only at 17 weeks, the time point at which dystrophin protein was barely detectable in the heart, and somewhat decreased in the diaphragm and quadriceps. A similar pattern was observed for circulating levels of aspartate aminotransferase and alanine aminotransferase (Figure 5b), which are present in a variety of extrahepatic tissues, including cardiac and skeletal muscles confirming that the restored dystrophin protein improved the integrity of muscle sarcolemma. Specifically to the heart, cardiac muscle stained with hematoxylin and eosin showed significant reduction of inflammatory cell infiltration in mdx mice treated with M23D-B at 3 weeks after the last injection (Figure 5c), suggesting that necrosis in cardiac muscle was significantly reduced.Figure 5Improvement of pathology markers in mdx mice treated intravenously with M23D-B at 12 mg/kg/day for 4 days. (a) Serum creatinine kinase (CK). C57BL (n = 5), treated mdx mice (n = 2 at each time point), and untreated mdx mice (n = 20). (b) Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). C57BL (n = 3), treated mdx mice (n = 2 at each time point), and untreated mdx mice (n = 10). All bar graphs represent mean except the bar graphs of serum CK of C57BL and untreated mdx mice, which represent mean and SEM. (c) Hematoxylin and eosin staining of cardiac muscle. C57BL at 8 weeks of age, treated mdx at 11 weeks of age (3 weeks after last injection), and untreated mdx at 8 weeks of age. Arrows, inflammatory infiltrate; Scale bar, 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionIn this study, we first used EGFP-654 mice to evaluate the potential of peptide-conjugated PMOs as splice-switching oligonucleotides. This mouse model provides a positive readout for functional biodistribution data, indicating where the conjugates are functionally active. The results clearly show that systemically delivered PPMOs corrected EGFP-654 splicing to a high degree in a number of different tissues, and were essentially nontoxic at the doses used in the study. Comparison with an unconjugated PMO that was investigated in previous experiments in the same animal model indicates that the CPPs altered the functional biodistribution of PPMOs and significantly enhanced their potency and intracellular delivery.4Sazani P Gemignani F Kang SH Maier MA Manoharan M Persmark M et al.Systemically delivered antisense oligomers upregulate gene expression in mouse tissues.Nat Biotechnol. 2002; 20: 1228-1233Crossref PubMed Scopus (269) Google Scholar The effects of the CPPs are dependent on the number and position of the non-α amino acids, aminohexanoic acid (X), and/or β-alanine (B) (X and/or B residues). These differences could be attributable to the stability of the conjugates in serum and cells,11Youngblood DS Hatlevig SA Hassinger JN Iversen PL Moulton HM Stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells.Bioconjug Chem. 2007; 18: 50-60Crossref PubMed Scopus (140) Google Scholar or to their ability to escape from endosomes/lysosomes.12Abes S Moulton HM Clair P Prevot P Youngblood DS Wu RP et al.Vectorization of morpholino oligomers by the (R-Ahx-R)4 peptide allows efficient splicing correction in the absence of endosomolytic agents.J Control Release. 2006; 116: 304-313Crossref PubMed Scopus (164) Google ScholarIn EGFP-654 mice, PPMO-B delivered by IV injection at 12 mg/kg/day for 4 days was highly potent in splicing correction in cardiac muscle, diaphragm, and quadriceps, and its activity persisted in these muscles for at least 2–3 months. Therefore, we applied its equivalent, PPMO M23D-B, in the treatment of muscular dystrophy in mdx mice. Systemic delivery of M23D-B induced skipping of exon 23, thereby restoring and sustaining dystrophin protein expression which localized correctly at the plasma membrane of muscle fibers in body-wide muscles, including the heart. It is also important to note that the smooth muscles of the digestive tract are impaired in DMD;26Barohn RJ Levine EJ Olson JO Mendell JR Gastric hypomotility in Duchenne's muscular dystrophy.N Engl J Med. 1988; 319: 15-18Crossref PubMed Scopus (130) Google Scholar PPMO-B has an effect on these tissues, e.g., stomach, small intestine, and colon (Supplementary Figure S1a), and should therefore provide amelioration of related symptoms in patients.Induction of exon 23 skipping mediated by M23D-B conjugate was significantly more fficient than that mediated by the unconjugated PMO.19Alter J Lou F Rabinowitz A Yin H Rosenfeld J Wilton SD et al.Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology.Nat Med. 2006; 12: 175-177Crossref PubMed Scopus (431) Google Scholar More importantly, the M23D-B showed induction of exon skipping, production of induced dystrophin protein, and histological improvement in cardiac muscle, the first such results using an antisense oligonucleotide. Given that 30% of deaths in DMD patients are caused by cardiomyopathy,27Foster K Foster H Dickson JG Gene therapy progress and prospects: Duchenne muscular dystrophy.Gene Ther. 2006; 13: 1677-1685Crossref PubMed Scopus (61) Google Scholar it is crucial to induce dystrophin expression in cardiac muscle. Clearly, the expression of dystrophin ameliorated the sarcolemmal integrity of muscles, leading to dramatic decreases in serum CK, aspartate aminotransferase, and alanine aminotransferase, and reduced the extent of muscle necrosis in the heart.Materials and MethodsPPMO conjugates. The sequences of CPPs are indicated in Table 1. All PPMOs were synthesized and purified as described by Wu et al.8Wu RP Youngblood DS Hassinger JN Lovejoy CE Nelson MH Iversen PL et al.Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity.Nucleic Acids Res. 2007; 35: 5182-5191Crossref PubMed Scopus (94) Google Scholar and targeted to the aberrant 5'-splic