The flow of genetic information begins with the DNA blueprint, which is transcribed into pre-messenger RNA (pre-mRNA). This pre-mRNA must undergo splicing before it can be translated into a functional protein. Splicing removes non-coding sections (introns) and precisely joins the coding sections (exons). Exon skipping is a therapeutic strategy that leverages this natural cellular process to correct specific genetic errors. By intentionally altering how the pre-mRNA is spliced, this method aims to produce a modified but usable protein, bypassing the damage caused by the original gene mutation.
The Molecular Mechanism of Exon Skipping
A gene’s instruction is read in three-nucleotide units called codons, which establish a “reading frame” that dictates the sequence of amino acids in the resulting protein. Many severe genetic diseases are caused by mutations, such as deletions, that remove a number of nucleotides not divisible by three. This error shifts the entire reading frame downstream of the mutation, leading to a garbled message. This typically results in a premature stop codon, which produces a severely truncated and non-functional protein.
The goal of exon skipping is to correct this disrupted reading frame at the pre-mRNA level. This is accomplished by causing the cellular splicing machinery to exclude a specific, targeted exon from the final messenger RNA (mRNA) transcript. While removing an exon might seem counterintuitive, the removal is strategically planned to restore the reading frame balance. When the exon upstream of the targeted exon is joined to the exon downstream, the resulting combined sequence is readable in three-nucleotide increments.
This restored reading frame allows the cell to continue protein production past the original mutation site, avoiding the premature stop signal. The resulting protein will be shorter because a section of its genetic code has been deliberately omitted. Despite its missing segment, this internally deleted protein is often partially functional. This is a significant improvement over the complete absence of the protein that characterizes the severe form of the disease. This correction essentially turns a severe mutation into a milder one that maintains some degree of protein function.
Tools Used to Induce Exon Skipping
The ability to induce this precise change relies on synthetic molecules called Antisense Oligonucleotides (ASOs). An ASO is a short, custom-designed strand of nucleic acid chemically synthesized to be complementary to a specific sequence within the pre-mRNA. The design is highly specific, ensuring the ASO binds only to its intended target on the pre-mRNA strand.
Once administered, the ASO enters the cell nucleus and physically binds to the pre-mRNA sequence that marks the beginning or end of the targeted exon. This binding acts like a molecular patch, masking the splice recognition signals the cell’s natural splicing machinery uses to identify and incorporate the exon. Because these signals are blocked, the splicing machinery is forced to skip over the masked exon and connect the adjacent exons instead.
To be effective, ASOs must overcome several biological hurdles. They require chemical modifications, such as those found in phosphorodiamidate morpholino oligomers (PMOs) or 2′-O-methyl phosphorothioate (2’OMePS) chemistries, to increase their stability and resistance to degradation by cellular enzymes. A challenge remains in ensuring efficient delivery of the ASOs into the target cells, particularly muscle cells. Furthermore, high concentrations must be achieved within the nucleus where splicing occurs to force the desired skipping event.
Clinical Application and Disease Targeting
Exon skipping has achieved its most significant clinical progress in treating Duchenne Muscular Dystrophy (DMD). DMD is a progressive muscle-wasting condition caused by mutations in the large dystrophin gene. DMD typically results from frameshift mutations that prevent the production of functional dystrophin, a protein crucial for muscle fiber stability. Restoring even a partially functional version of this protein can significantly lessen the disease severity.
The most common therapeutic strategy targets exon 51 of the dystrophin gene, applicable to approximately 13% of all DMD patients who have specific deletion patterns. By skipping this exon, the out-of-frame mutation is corrected, allowing the production of a truncated but functional dystrophin protein. This resulting protein is similar to that found in the milder condition, Becker Muscular Dystrophy. This approach does not cure the disease but aims to slow the progression of muscle deterioration.
Several ASO treatments based on this technology have received regulatory approval from the U.S. Food and Drug Administration (FDA). These include eteplirsen (targeting exon 51), golodirsen (targeting exon 53), and casimersen (targeting exon 45). These approved therapies are highly mutation-specific, meaning a patient must have a mutation that is “amenable” to the skipping strategy to be effective. The success of these therapies has paved the way for developing similar ASOs to target other exons, broadening the scope of this precision medicine approach.