A renaissance for antisense oligonucleotide drugs in neurology: exon skipping breaks new ground

Abstract

Antisense oligonucleotides are short nucleic acid sequences designed for use as small-molecule drugs. They recognize and bind to specific messenger RNA (mRNA) or pre-mRNA sequences to create small double-stranded regions of the target mRNA that alter mRNA splicing patterns or inhibit protein translation. Antisense approaches have been actively pursued as a form of molecular medicine for more than 20 years, but only one has been translated to a marketed drug (intraocular human immunodeficiency virus treatment). Two recent advances foreshadow a change in clinical applications of antisense strategies. First is the development of synthetic DNA analogues that show outstanding stability and sequence specificity yet little or no binding to modulator proteins. Second is the publication of impressive preclinical and clinical data using antisense in an exon-skipping strategy to increase dystrophin production in Duchenne muscular dystrophy. As long-standing barriers are successfully circumvented, attention turns toward scale-up of production, long-term toxicity studies, and the challenges to traditional drug regulatory attitudes presented by tightly targeted sequence-specific drugs.

Figure 1

Comparison of chemistries used for the exon-skipping approach. Examples of artificially developed antisense oligomers such as 2′-O-methylated antisense oligonucleotides (2′-O-MeAO) (phosphorothioate), locked nucleic acid (LNA), phosphorodiamidate morpholino oligomers (PMOs), and peptide-tagged PMOs (PPMOs) are shown for comparison with DNA and RNA.

Figure 2

Clinical phenotypes associated with specific start (A) and end (B) sites for in-frame deletions. Percentages of patients with Duchenne muscular dystrophy (DMD) out of patients with DMD or Becker muscular dystrophy with specific start and end exons are shown. Combined muscular dystrophy databases of 14 countries (from Argentina, Belgium, Brazil, Bulgaria, Canada, China, Denmark, France, India, Italy, Japan, The Netherlands, the United Kingdom, and the United States) at Leiden University (http://www.dmd.nl), where diagnoses were performed using multiplex ligation-dependent probe amplification/multiplex amplification and probe hybridization, Southern blotting, or polymerase chain reaction primer sets that allow deletion boundaries to be assigned accurately to a specific exon, are used (deletion start sites: n=288 for exon 45, n=23 for exon 47, n=9 for exon 48, n=12 for exon 49, and n=10 for exon 50; deletion end sites: n=11 for exon 46, n=115 for exon 47, n=95 for exon 48, n=51 for exon 49, n=53 for exon 51, n=40 for exon 53, and n=21 for exon 55).

Figure 3

Targets of exon skipping and population of potentially treatable patients. Percentage of patients with the dystrophin deletion who are potentially treatable by targeting specific exons for Duchenne muscular dystrophy. For example, 17% of patients with Duchenne muscular dystrophy who have the dystrophin deletion can be potentially treated by targeting exon 51 using antisense oligonucleotides.

Figure 4

Mechanism of multiexon skipping of exons 45 through 55 to rescue 60% of patients with Duchenne muscular dystrophy with dystrophin deletions A, More than 60% of deletion mutations of the dystrophin gene occur within the hot-spot range of exons 45 through 55 (exon 45 is deleted in this schematic [del]) in Duchenne muscular dystrophy muscles. The messenger RNA (mRNA) of remaining exons is spliced together but the reading frame is disrupted, resulting in failure of the production of functional dystrophin protein. CK indicates creatine kinase; Ca²⁺, calcium ions. B, An antisense oligonucleotide (AO) cocktail targeting exons 45 through 55 likely enters the Duchenne muscular dystrophy muscle through its leaky membranes, then binds to the dystrophin mRNA in a sequence-specific manner. The AOs block the splicing machinery and prevent inclusion of all exons between exons 45 and 55. Skipping these exons restores the reading frame of mRNA, allowing production of quasi-dystrophin containing exons 1 through 44 and exons 56 through 79, which is not normal but likely retains considerable function as evidenced by patients with clinically milder Becker muscular dystrophy with identical partial dystrophin.