In their nucleic acid base sequences, RNA and DNA encode the sequences of amino acids that make up proteins.  In cells with nuclei (including human cells) RNA is copied from DNA in the nucleus and the RNA is exported to the cytoplasm, where it binds to ribosomes.  A ribosome uses the information in an RNA (the RNA’s base sequence) to direct the assembly of a protein by specifying the order of its constituent amino acids.  The DNA serves as long-term information storage, the RNA serves as temporary copies of that information, and the proteins provide structure, catalysis, signaling, and many other contributions to keep a living cell functioning.

While RNAs are still in the nucleus, some sections (they are called introns) are removed from most RNAs and the remaining sections (they are called exons) are ligated together.  This process is mediated by a protein-RNA complex called a spliceosome.  Early on, while the introns are still within an RNA, it is called a pre-mRNA.  Once the introns have been removed and a few other processing steps are completed (capping and tailing) the RNA is called a messenger RNA (mRNA).  The process of removing the introns is called RNA splicing.  Splicing is regulated in cells and different exons are included in mRNAs depending on what specific tissue the RNA is made in or what stage of embryonic development the cells are undergoing.  This alternative splicing is controlled by the binding of splice-regulatory proteins to particular sites on pre-mRNA.  The mix of active splice-regulatory proteins present in the nucleus varies by tissue type and developmental stage.  Alternative splicing allows a single gene to be used to produce several different proteins by using different sets of exons to make different mRNAs based on a single DNA sequence.

RNA splicing can be altered in the lab or clinic using a particular class of antisense oligos, called steric-blocking oligos.  Antisense oligos have sequences of bases usually about 15 to 30 bases long, with bases similar (and usually identical) to nucleic acids.  The oligos bind to complementary sequences of RNA.  Steric-blocking oligos have unnatural backbones, different from the ribose-phosphate or deoxyribose-phosphate backbones of RNA or DNA respectively.  What sets steric-blocking oligos apart from other antisense types is that they do not activate enzymes which degrade RNA, while other antisense types can do this.  Because they bind RNA but do not cause the RNA they are bound with to be degraded, steric-blocking oligos are used to get in the way of other molecules, denying them access to a site on an RNA but leaving the RNA otherwise intact.  Some examples of steric-blocking antisense are 2’-O-methyl phosphorothioate oligos, 2’-O-methoxyethyl phosphorothioate oligos, locked nucleic acids, peptide nucleic acids, Morpholino oligos, and many other antisense structural types.  We’ll focus on one member of the steric-blocking antisense class, the Morpholinos, oligos both widely used in research and with examples approved as drugs.

Morpholino oligos are widely used in biological research to change the patterns of splicing of pre-mRNAs, producing altered mRNAs.  Splice modification is almost always done using one of two methods: commonly (1) by blocking the proteins and RNA-protein complexes from binding near the splice junctions to direct the spliceosome where to cut out introns, or sometimes (2) by binding the sites where splice regulatory proteins would otherwise bind RNA, interrupting a signal directing alternative splicing.  Embryos are very sensitive to changes in the biochemistry within their cells.  Independent of binding to RNA, the Morpholino oligos have little effect on cells.  Because they bind a target RNA sequence but do little else, Morpholinos are often microinjected into embryos of model organisms including zebrafish, African clawed frogs, sea urchins, sea anemones and many others.  By altering or eliminating the activity of a targeted protein, investigators learn about the function of the targeted protein during embryonic development.  Morpholinos are also used for research in adult organisms, organoids, explants and cell cultures.

As of 2024, four Morpholino oligos have been approved by the US Food and Drug Administration (FDA) as drugs for treatment of some mutated alleles causing Duchenne muscular dystrophy (DMD), a disease which involves degeneration of muscle and slow loss of muscle function.  DMD is caused by new or inherited mutations in the gene encoding the human dystrophin protein.  The dystrophin gene is on the X sex chromosome, so most cases appear in males, who have only a single X chromosome and so a single dystrophin allele.  For some frameshifting mutations causing DMD, altering splicing of pre-mRNA with a steric-blocking antisense oligo can produce an mRNA that directs synthesis of a functional protein.  This process involves using an oligo to splice out (“skip”) an exon when making the mRNA.  This does not make the same dystrophin protein present in a person who does not have DMD; the protein produced from an oligo-treated RNA will be shorter than the protein made by a person without oligo treatment.  Oligos have been found to offer some benefit to some people who cannot otherwise make functional dystrophin.

One approved Morpholino oligo targets at a splice junction to alter splicing (casimersen), while the other approved drugs target splice regulatory protein binding sites.  All of these are frameshifting targets (explained in the next paragraph), each intended to treat a specific group of frameshift mutations. 1. eteplirsen (EXONDYS 51) targets exon 51; Sarepta Therapeutics Inc. 2. golodirsen (VYONDYS 53) targets exon 53; Sarepta Therapeutics Inc. 3. casimersen (AMONDYS 45) targets exon 45; Sarepta Therapeutics Inc. 4. viltolarsen (VILTEPSO) targets exon 53; NS Pharma Inc. (subsidiary of Nippon Shinyaku Co., Ltd.) Exons are numbered as for the human dystrophin transcript Dp427 m, the most common muscle transcript.

As an mRNA passes through a ribosome, bases are read in groups of three. Each three-base group, called a “codon”, specifies either a particular amino acid or a signal for the ribosome to stop (a stop codon).  The positions where the long string of RNA bases is ruled into the three-base groups is called the reading frame.  It is the ribosome moving in three-base steps along the RNA that determines the reading frame; the mechanism is fascinating, involving transfer RNAs (tRNA), but I won’t describe it here.  A genetic code chart shows how each codon is related to a single amino acid or a stop.  If a ribosome was to slip one extra base forward as it makes a string of amino acids, moving four bases ahead instead of three, the groups subsequently read (the “downstream” codons) would no longer be the same three-base groups as the un-slipped ribosome would have encountered; the breaks between the codons would have slipped to be between different bases. This change in the codons read by the ribosome is called a frameshift and results in changing the amino acid sequence of the part of the protein assembled after the ribosome has made the single-base slip.  Similarly for a DNA change (mutation), if a single DNA base is added or removed, or if two bases are added or removed from the DNA sequence, then when that DNA is copied into RNA and goes to the ribosome the downstream codon groups will fall in the wrong places on the sequence.  The amino acid sequence produced will be changed from that mutated sequence onward downstream; this is called a frameshift mutation. However, if three bases are removed from the DNA encoding the amino acid sequence of a functional protein then, when the corresponding RNA goes to the ribosome, all the downstream codons are read in the correct reading frame.  This produces a downstream amino acid sequence which is the same as in the original functional protein (though one amino acid is missing) and so this three-base deletion of DNA is not a frameshift mutation.

A deletion of an exon that skips several bases which is evenly divisible by three will not change the reading frame of the downstream sequence; such an exon is called a cassette exon.  Cassette exons are commonly included or deleted during alternative splicing, usually producing alternative functional forms of a protein.  Deletion of an exon which skips several bases that is not evenly divisible by three will alter the reading frame of the downstream sequence and will alter the amino acid sequence produced on the ribosome from the frameshifted sequence.  This sort of deletion can cause the loss of most or all a protein’s function.

Here’s a simple illustration of frameshifting.  Consider this English sentence of three letter words, always breaking the words into three letter groups: The fat red cat ate the big rat and sat If you remove one letter and still use groups of three letters, you get: The fat rec ata tet heb igr ata nds at Which makes no sense on its downstream end.  Remove two more letters: The fat cat ate the big rat and sat You haven’t restored all the meaning, but at least the downstream words make sense.

So far, therapeutic Morpholinos targeting the dystrophin gene cause exons to be skipped, that is, cause exons to be excluded from the mature mRNA.  Typically, oligos are used to treat people who have deletion mutations in their dystrophin gene that cause the RNA sequence downstream of the deletion to be frameshifted as it passes through the ribosome, producing a string of amino acids very different from what is needed to make functional dystrophin.  The oligos approved so far are each designed to bind to an exon adjacent to a frameshift deletion, skip the exon, and so cause another shift of the reading frame, correcting the frameshift caused by the original deletion.  This restores the reading frame downstream so that functional (though internally shortened) dystrophin can be made.  So, a frameshifting mutation can potentially have its reading frame corrected by skipping an adjacent exon to produce another frameshift; that is the technique used with the currently approved Morpholino oligos for treating DMD.  A specific mutation must be matched to the right oligo, and so far the four approved oligos for treating DMD can only treat a fraction of the DMD population; FDA approval of more oligo sequences will allow treatment of other mutations.

Much of the early development of therapeutic oligos for DMD was based on research using the mdx mouse model, a mouse with a mutation that created a new stop codon in exon 23 of its dystrophin gene.  With the ribosome halting at a stop codon in exon 23, the downstream codons are not ready to make a longer dystrophin protein.  The truncated protein which is produced from the sequence upstream of the premature stop codon is not enough to fulfill the requirements of a healthy muscle cell.  Exon 23 of mouse dystrophin is a cassette exon, an exon with several bases evenly divisible by three.  Skipping that exon with a steric-blocking oligo removes the stop codon and leaves the downstream sequence in the useful reading frame.  That means the dystrophin protein produced using an mdx mouse transcript without exon 23 will be assembled from the correct amino acids downstream of the skipped exon.  When Morpholino oligos causeexon 23 to be skipped in mdx mice, they make a functional form of dystrophin.  While many people with DMD have frameshifts in the DMD gene, some have premature stop codons instead.  In humans, there is the potential to treat premature stop codons occurring in cassette exons with single steric-blocking oligo sequences targeting those exons.

Many other genetic diseases may be treatable by steric-blocking oligos altering pre-mRNA splicing.  Many proof-of-principle studies have demonstrated oligo efficacy for altering splicing of RNAs carrying other disease-causing mutations.  A current weakness of these antisense approaches for therapy is that delivery from blood into cells is inefficient.  New compounds, in which cell-penetrating peptides or antibodies or antibody fragments are bound to oligos, have worked well in preclinical studies and are entering clinical trials.  These might provide the improved cell delivery needed for more widespread application of oligos for treating human genetic diseases.  Once nontoxic and effective delivery of therapeutic oligos has been proven, oligos might be employed for the treatment of a broad range of previously intractable human genetic conditions.