Splicing: Hear and Now!
Authors: Ryszard Kole
Journal: Molecular Therapy (2013)

Approximately 1 in 1,000 babies is born with hereditary loss of hearing, translating into 40,000 newborns affected by deafness in the United States alone. The disease is due primarily to genetic defects leading to degeneration of hair cells in the inner ear that convert sound waves into electrical signals and of the auditory neurons that carry those signals to the brain. A frequent (1 in 6,000 births) and severe consequence of such defects is Usher syndrome, in which deafness may be combined with blindness and balance dysfunction. In humans, hair cells and auditory neurons are generated only during fetal development, and in affected individuals these sensory cells are lost soon after birth.1Conde de Felipe MM Feijoo Redondo A García-Sancho J Schimmang T DuránAlonso MB Cell- and gene-therapy approaches to inner ear repair.Histol Histopathol. 2011; 26: 923-940PubMed Google Scholar It is therefore quite a feat that researchers, led by Michelle Hastings from Rosalind Franklin University, were able to restore hearing and balance in a mouse model of Usher syndrome. It is even more remarkable that this feat was accomplished with a single injection of an antisense oligonucleotide (ASO) and that the therapeutic effects persisted for several months.2Lentz JJ Jodelka FM Hinrich AJ McCaffrey KE Farris HE Spalitta MJ et al.Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness.Nat Med. 2013; 19: 345-350Crossref PubMed Scopus (152) Google Scholar Usher syndrome is caused by mutations in the USH1C gene, which encodes harmonin, a protein that plays a role in proper development of hair cells. As is common in inherited disorders, a large number of mutations can affect USH1C gene expression. One causative mutation, USH1C216G>A, creates an aberrant 5′ splice site in exon 3 of the harmonin pre-messenger RNA (pre-mRNA) that is preferentially used, leading to incorrect splicing of the RNA and preventing proper translation of harmonin protein (Figure 1). In the new study, Hastings’ group screened a series of ASOs targeted to exon 3 of the USH1C216G>A pre-mRNA and identified an effective ASO (ASO-29) that blocked usage of the aberrant splice site, thereby restoring correct harmonin mRNA splicing and protein translation in cultured cells.2Lentz JJ Jodelka FM Hinrich AJ McCaffrey KE Farris HE Spalitta MJ et al.Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness.Nat Med. 2013; 19: 345-350Crossref PubMed Scopus (152) Google Scholar Intraperitoneal injection of newborn mice (216AA; carrying the human USH1C216G>A gene) with 300 mg/kg of ASO-29 eliminated the unusual circling behavior (a symptom of loss of balance) of treated mice. There was no improvement in mice treated with control ASOs. The improvement was seen after a single injection of ASO-29 up to postembryonic day 13 but not in mice injected on day 16. Clearly the temporal window of opportunity to treat the mice is very short (see also below). In the rescued mice, the improvements in circling behavior persisted for at least 8 months. Equally impressive was the improvement in hearing of ASO-29–treated 216AA mice. These mice responded to loud sounds with a startle followed by a freezing response, whereas the untreated 216AA mice did not respond to this stimulus. Quantitative analyses of brainstem waveforms stimulated by the sound stimuli showed responses to low- and mid- but not high-frequency sound, indicating that hearing restoration was not complete. These quantitative measurements also showed slow deterioration of the response, although even at 6 months posttreatment the waveform response was above that of the control mice or 216AA mice treated with control ASOs. Finally, a series of other analyses showed that, as could be expected, the underlying molecular mechanisms were also corrected—that is, the levels of correctly spliced harmonin mRNA and full-length harmonin protein were elevated over background in untreated or control-treated mice. Moreover, the protein was properly localized to the stereocilia of hair cells. Interestingly, the level of correct splicing of USH1C216G>A pre-mRNA, even at its highest level at day 120 after birth, was only about 5% of normal, whereas the level of harmonin protein reached about 50% of normal and was roughly equivalent to the harmonin level in a heterozygote Ush1c mouse. As mentioned above, individuals with Usher syndrome present with loss of both hearing and vision, and both impairments are recapitulated in the 216AA mice. However, in these mice the retinitis pigmentosa symptoms appear only about one year after birth, and they were not provided in this paper. Several interesting observations stem from this work. The ASO used in the study (an 18-mer oligoribonucleotide) was chemically modified with a phosphorothioate backbone and 2′-O-methoxyethyl residues across the whole sequence (2′OMOE PS). These chemical modifications increase druglike properties of natural nucleic acids (RNA or DNA) by enhancing their resistance to degradation in the bloodstream and improving uptake into target cells. In a previous study, this type of ASO was found to have an elimination half-life from mouse liver of 20 days, and its inhibitory effects on the expression of the target mRNA, apoB, were no longer measurable 8 weeks after last dosing with the compound.3Yu RZ Lemonidis KM Graham MJ Matson JE Crooke RM Tribble DL et al.Cross-species comparison of in vivo PK/PD relationships for second-generation antisense oligonucleotides targeting apolipoprotein B-100.Biochem Pharmacol. 2009; 77: 910-919Crossref PubMed Scopus (97) Google Scholar Because in the study by Hastings’ group the effects of ASO-29 could be detected 6 months posttreatment, it appears that the 2′OMOE PS ASO is more stable in hair cells than in liver cells. Another distinction is that the ASO in this study was used to target a splicing intermediate in the nucleus, as opposed to cytoplasmic mRNA as in the previous study. A further implication is that the ASO may be more stable in the nucleus than in the cytoplasm. If the same holds true in human cells, dosing of patients could be very infrequent, which would be beneficial for treatment of a chronic condition. An advantage of hearing loss as a disease target is that the therapeutic intervention does not need to be systemic. One can expect that injections into the ear or, even more specifically, the cochlea may be sufficient to deliver the nucleic acid drug to targeted cells. A significant advantage of this delivery route is that any negative side effects—an important consideration for systemic delivery of nucleic-acid based drugs—should be minimized. In addition, the required dose should be very small, thereby reducing the cost of lifelong treatment. Macugen (pegaptanib), an intravitreally injected RNA aptamer for treatment of macular degeneration, is an example of a drug that meets the criteria of not requiring systematic administration or a large dose4Zehetner C Kirchmair R Huber S Kralinger MT Kieselbach GF Plasma levels of vascular endothelial growth factor before and after intravitreal injection of bevacizumab, ranibizumab and pegaptanib in patients with age-related macular degeneration, and in patients with diabetic macular oedema.Br J Ophthalmol. 2013; 97: 454-459Crossref PubMed Scopus (135) Google Scholar. The data from the study by Hastings’ group show that the injected drug was effective up to day 13 after birth but not at day 16. One hopes that this is because at the latter time point the targeted hair cells had already degenerated beyond repair and not because more mature cells do not take up the oligonucleotide. This is an important caveat of the present study, which, fortunately, can be easily addressed through continued experimentation with repeated injections. If, as is likely, the rescued adult cells continue responding to treatment with ASO-29, it will be possible to demonstrate that the splicing modulation approach also relieves blindness in individuals with Usher syndrome. The data also show that restoration of as little as 5% of correct mRNA splicing generates about 50% of desired harmonin protein and restores sufficient hearing and mobility to the treated mice. This illustrates the power of modulation of splicing and its advantages over small interfering RNA and classic antisense approaches designed to inhibit expression of the target gene. In this approach, reduction of expression of a viral gene or oncogene target mRNA by 5% or protein by 50% is unlikely to be clinically relevant. As is common in genetic disorders, a large number of different gene mutations can be responsible for the disease. In this context, the drawback of oligonucleotide-based RNA splicing modulation is that the mechanism is sequence- and mutation-specific, and consequently the potential ASO-29 treatment is applicable to only 6–8% of Usher patients. Other treatment mechanisms will need to be explored to help the remaining patients with this syndrome. The first use of oligonucleotides to induce splicing modulation was as a potential treatment for β-thalassemia, in which—similarly to the present study—a splicing mutation was blocked and correct splicing of β-globin was restored.5Dominski Z Kole R Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides.Proc Natl Acad Sci USA. 1993; 90: 8673-8677Crossref PubMed Scopus (335) Google Scholar Even then it was evident that this approach may have much broader application. Indeed, a large number of diseases should be treatable with this technology, and the effective mechanisms include not only correction of splicing defects but also manipulation of alternative splicing or generation of novel splice variant proteins.6Kole R Krainer AR Altman S RNA therapeutics: beyond RNA interference and antisense oligonucleotides.Nat Rev Drug Discov. 2012; 11: 125-140Crossref PubMed Scopus (852) Google Scholar To date, the most advanced applications of oligonucleotide-induced modulation of RNA splicing are for treatments for Duchenne muscular dystrophy7Cirak S Arechavala-Gomeza V Guglieri M Feng L Torelli S Anthony K et al.Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study.Lancet. 2011; 378: 595-605Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar,8Goemans NM Tulinius M van den Akker JT Burm BE Ekhart PF Heuvelmans N et al.Systemic administration of PRO051 in Duchenne's muscular dystrophy.N Engl J Med. 2011; 364: 1513-1522Crossref PubMed Scopus (595) Google Scholar and spinal muscular atrophy9ClinicalTrials.gov An open-label safety, tolerability and dose-range finding study of multiple doses of ISIS SMNRx in patients with spinal muscular atrophy SMNRx—CS2 (NCT01703988).http://www.clinicaltrials.gov/ct2/show/NCT01703988?term=SMA&rank=26Google Scholar that are undergoing clinical trials. It bodes well for the field that novel indications, such as the one described by Hastings’ group, are not far behind.