Peptide-Conjugated Antisense Therapy Takes a Skip Ahead
Authors: Dominic J Wells
Journal: Molecular Therapy (2008)

Antisense-mediated exon-skipping is a potential treatment for the lethal muscle wasting condition Duchenne muscular dystrophy (DMD). Local treatment in the dystrophic mdx mouse model leads to efficient restoration of dystrophin expression. Antisense can also be delivered systemically but requires repeated administration and high doses to achieve a therapeutically useful effect. The article by Jearawiriyapaisarn and colleagues1Jearawiriyapaisarn N Moulton HM Buckley B Roberts J Sazani P Fucharoen S et al.Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice. 2008; (Mol Ther 16: 1624–1629.)Google Scholar in this issue of Molecular Therapy addresses the problem of low efficiency of systemic delivery by coupling an arginine-rich cell-penetrating peptide (CPP) to a phosphorodiamidate morpholino oligomer (PMO). Treatment for 4 days with low doses of this conjugate lead to highly efficient and prolonged exon skipping and the production of therapeutically useful levels of dystrophin in all skeletal muscles, as well as the heart. These results promise a significantly improved reagent for human clinical trials. Antisense RNA is most commonly used as a method for blocking protein production. Delivery of single-stranded antisense oligonucleotide (ON) targeting a specific messenger RNA (mRNA) with subsequent recognition of the RNA-ON duplex by RNase H leads to RNA destruction. More recently, attention has been focused on RNA interference, in which short double-stranded RNA duplexes are recognized by the cellular RNA–induced silencing complex (RISC). The small interfering RNA duplex is unwound by a helicase within RISC; one (guide) strand remains within the complex while the passenger strand is degraded. The activated complex then mediates the cleavage of mRNA species that interact with the RISC-associated guide strand. The disadvantage with these techniques is that the ONs are susceptible to metabolism and so need to be delivered on a frequent basis or by a viral or nonviral vector with all the attendant issues surrounding the efficiency of delivery and interactions with the immune system. Significant changes in the chemistry that promote increased stability of ONs decrease the ability to activate RNase H or the RISC complex. An alternative approach is to use chemical modifications that substantially prevent metabolism of the ON and utilize the steric blockage that arises when these stable molecules bind to the target RNA sequence. Such molecules can be used to block the translation start site of an mRNA or to prevent binding of regulatory microRNAs to the 3′ untranslated region of the mRNA. A further application of this steric blockade is to modulate the splicing of exons from the primary transcript. By binding to the splice sites at one end or the other of an exon, or to one of the exon recognition sequences, specific exons can be masked from the splicing machinery and so be omitted from the final mRNA. It is this latter use that has been proposed as a treatment for DMD. The majority of mutations in individuals with DMD involve a disruption of the open reading frame, which leads to the failure to produce a stable form of the dystrophin protein. Without this protein, muscle fibers are very easily damaged and the cycles of necrosis and repair not only exhaust the regenerative capacity of muscle but also cause extensive fibrosis, both features leading to loss of muscle function. The milder allelic condition, Becker muscular dystrophy, is commonly associated with mutations that maintain the reading frame and hence the production of an internally deleted but still semifunctional protein. Some individuals with Becker muscular dystrophy do not develop disability until late in life, and so the restoration of the reading frame by deletion of one or more additional exons at the site of mutation offers the prospect of a considerable improvement in phenotype for persons with DMD. Antisense-induced exon-skipping was first demonstrated in lymphocytes of a DMD patient and in muscle cells of the mdx mouse and has subsequently been extensively studied in human and animal cells in vitro and the mdx mouse in vivo (e.g., refs. 2Pramono ZA Takeshima Y Alimsardjono H Ishii A Takeda S Matsuo M Induction of exon skipping of the dystrophin transcript in lymphoblastoid cells by transfecting an antisense oligodeoxynucleotide complementary to an exon recognition sequence.Biochem Biophys Res Commun. 1996; 226: 445-449Crossref PubMed Scopus (111) Google Scholar,3Dunckley MG Manoharan M Villiet P Eperon IC Dickson G Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides.Hum Mol Genet. 1998; 7: 1083-1090Crossref PubMed Scopus (207) Google Scholar,4Wilton 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 (181) Google Scholar,5van Deutekom JC Bremmer-Bout M Janson AA Ginjaar IB Baas F den Dunnen JT et al.Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells.Hum Mol Genet. 2001; 10: 1547-1554Crossref PubMed Scopus (246) Google Scholar,6Mann 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 (339) Google Scholar). The mdx mouse is a commonly used biochemical model of DMD. Although the mdx shows few clinical signs until very late in life, it lacks dystrophin and undergoes cycles of muscle degeneration and regeneration. The failure to produce dystrophin is due to a premature stop mutation in exon 23 of the murine dystrophin gene, and removal of this exon restores the reading frame and thus the production of dystrophin. A single intramuscular administration of antisense oligonucleotide (AO) targeting exon 23 leads to a high level of exon skipping and substantial restoration of dystrophin. However, to achieve similar results with systemic delivery requires repeated high-dose treatments, and no exon skipping is seen in the heart.7Alter J Lou F Rabinowitz A Yin H Rosenfeld J Wilton SD Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology.Nat Med. 2006; 12: 175-177Crossref PubMed Scopus (423) Google Scholar In the new study, Jearawiriyapaisarn and colleagues1Jearawiriyapaisarn N Moulton HM Buckley B Roberts J Sazani P Fucharoen S et al.Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice. 2008; (Mol Ther 16: 1624–1629.)Google Scholar addressed this issue of low efficiency by screening a range of CPPs to select the most efficient for delivery to skeletal and cardiac muscle using a transgenic mouse in which splicing of an intron inserted into the enhanced green fluorescent gene allowed expression of the reporter gene. Reverse transcriptase–polymerase chain reaction assessment of the percentage of spliced product and in vivo imaging were used to select the best peptide. The most promising candidate (PPMO-B) was then tested for its ability to deliver antisense targeting exon 23 to induce exon skipping in the mdx mouse. Treatment with just 12 mg/kg/day for 4 days led to very high exon skipping in the skeletal muscle with therapeutic levels of dystrophin for as long as 11 to 17 weeks, depending on the muscle. This was associated with a significant decline in serum creatine kinase, a whole-body indicator of increased muscle stability. As well as demonstrating high-efficiency delivery of the AO to the skeletal muscles and extensive exon skipping, the study demonstrated, for the first time, effective AO delivery and exon skipping in the mdx heart, which previously had been achieved only by using an adeno-associated viral vector to deliver antisense sequences.8Denti MA Rosa A D'Antona G Sthandier O De Angelis FG Nicoletti C et al.Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model.Proc Natl Acad Sci USA. 2006; 103: 3758-3763Crossref PubMed Scopus (112) Google Scholar Interestingly, a previous study using another CPP-conjugated PMO (peptide K in the present study) did not observe exon skipping in the mdx heart.9Fletcher S Honeyman K Fall AM Harding PL Johnsen RD Steinhaus JP et al.Morpholino oligomer-mediated exon skipping averts the onset of dystrophic pathology in the mdx mouse.Mol Ther. 2007; 15: 1587-1592Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar One of the major attractions of the PMO chemistry is that not only are the oligomers not metabolized (and hence they have a long period of action) but the backbone is uncharged and so they do not undergo nonspecific electrostatic interactions with other molecules. Consequently, they show very little toxicity both in preclinical testing and in clinical trials.10Amantana A Iversen PL Pharmacokinetics and biodistribution of phosphorodiamidate morpholino antisense oligomers.Curr Opin Pharmacol. 2005; 5: 550-555Crossref PubMed Scopus (112) Google Scholar In contrast, the toxicity of CPP-PMOs in humans is less well known, and this is even more of a concern when one considers that such molecules will need to be given repeatedly for many years if they prove successful in the treatment of DMD. The presence of the peptide raises the possibility of inducing immune responses in some individuals, and the charged amino acids may lead to interactions with other molecules. The results presented by Jearawiriyapaisarn and colleagues showed no evidence of acute toxicity in the mdx mouse, but studies with related CPPs have shown evidence of toxicity at high doses.11Amantana 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 (136) Google Scholar Chronic studies with repeated administration in nonhuman primates are likely to be required before use in humans. Thus, although the current results are exciting, it may be some time before these compounds are in regular clinical use. AOs are already in clinical trial for DMD. A single DMD patient was treated systemically with a phosphorothioate AO targeting exon 19 (ref. 12Takeshima Y Yagi M Wada H Ishibashi K Nishiyama A Kakumoto M Intravenous infusion of an antisense oligonucleotide results in exon skipping in muscle dystrophin mRNA of Duchenne muscular dystrophy.Pediatr Res. 2006; 59: 690-694Crossref PubMed Scopus (91) Google Scholar) with no adverse events but with limited success. More recently, intramuscular injection of a 2′O-methyl phosphorothioate AO targeting exon 51 was performed in four boys with DMD mutations corrected by exon 51 skipping. The results were impressive, with clear evidence of exon skipping and a substantial number of muscle fibers in the treated site expressing dystrophin that was also detectable on western blot.13van Deutekom JC Janson AA Ginjaar IB Frankhuizen WS Aartsma-Rus A Bremmer-Bout M et al.Local dystrophin restoration with antisense oligonucleotide PRO051.N Engl J Med. 2007; 357: 2677-2686Crossref PubMed Scopus (676) Google Scholar A similar study with a PMO targeting exon 51 is ongoing in the United Kingdom. Both groups have developed plans for systemic-delivery trials targeting exon 51, and the results will be awaited with interest.