Here are some types of oligos sorted by their mechanisms.
- Phosphorothioate DNA
RNase-independent (steric blocking oligos)
- 2’-O-substituted oligos (often phosphorothioate linkages)
- 2’-O methyl
- 2’-O methoxyethyl
- PNA (peptide nucleic acids)
- LNA (locked nucleic acid)
About the Morpholino drugs
The mechanism of the Morpholino drugs for Duchenne muscular dystrophy (DMD) is to induce skipping of an exon so that the downstream sequence is frameshifted. These are used to treat specific frameshift mutations of the human dystrophin gene, so that the frameshift induced by the oligo restores the correct reading frame to make the dystrophin protein. Frameshift mutations are either insertions or deletions in the DNA or splice-site mutations that cause insertions or deletions in the RNA. Typically these drugs have been used in the clinic to treat deletions, but research is ongoing to use oligos for treatment of insertions. The wild-type dystrophin has a reading frame that makes a functional protein, the mutation causes a frameshift so everything after the mutation has altered amino acid sequence, the oligo restores the reading frame so that the protein is made with a missing part somewhere in the middle but the downstream part of the protein has the same amino acid sequence as the protein made from the wild-type gene.
These are the Morpholino drugs currently US FDA approved for DMD (April 2021):
- Sarepta Therapeutics
eteplirsen EXONDYS 51 (SRP-4051) exon 51 dystrophin
golodirsen VYONDYS 53 (SRP-4053) exon 53 dystrophin
casimersen AMONDYS 45 (SRP-4045) exon 45 dystrophin
- NS Pharma (Nippon Shinyaku)
viltolarsen (NS-065/ NCNP-01) exon 53 dystrophin
There is an interesting oligo currently in clinical trials from Sarepta Therapeutics. This is SRPT-5051, which is a Morpholino with the same target as eteplirsen but with a cell-penetrating peptide attached. https://clinicaltrials.gov/ct2/show/NCT04004065
Blocking mutant splice sites
Instead of using a splice-modifying oligo to correct a reading frame, there is a different approach available for treating some mutations. Occasionally a mutation will create a new splice site. This can redirect splicing from the one of the splice sites used in the wild-type pre-mRNA to a new location in an exon or an intron. An early example of using an RNase-independent oligo to block a splicing site was the work by Ryszard Kole’s group with mutations causing beta-thalassemia, which is caused by defects in the human beta-globin gene (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC24007/). They worked with splice sites created by mutations and showed that by blocking the mutant splice site, splicing could be redirected to the wild-type splice site. Soon their early work with 2’-O-methyl phosphorothioate oligos was supplanted by Morpholino oligos and after a handful more papers Kole joined Sarepta Therapeutics (then called AVI BioPharma) where he participated in development of the DMD Morpholinos.
Another disease where research has been done with oligos blocking mutant splice sites to restore normal splicing is cystic fibrosis, caused by mutations in the CFTR gene. Morpholinos have been used for blocking mutant CFTR splice sites (for example, https://pubmed.ncbi.nlm.nih.gov/32520327/).
Upregulation with Morpholino oligos
Usually researchers think of antisense, and Morpholinos, as knockdown reagents. However, both of the treatments for mutations that I have described, reading-frame correction and blocking new splice sites created by mutations, are upregulation strategies that restore some or all of the function of a lost protein. A strategy we’ve not yet discussed can be used when an mRNA undergoes several splicing pathways, one leading to a functional protein and other(s) leading to splice forms with premature termination codons and undergoing nonsense-mediated decay (NMD); in some cases a Morpholino can be designed to shift the splicing away from the NMD mRNA to form more of the useful mRNA that makes a functional protein. Another upregulation strategy is to block the microRNA response element on the 3’-UTR of an mRNA, relieving that mRNA of translation suppression by an miRNA. Still another is offered when an intronic polyadenylation signal competes with splicing, so that full length mRNA is produced and truncated mRNA with an early poly-A tail is also produced; in this case, blocking the polyadenylation sequence with a Morpholino caused more of the full-length RNA to be made (https://www.nature.com/articles/nature20160).
Other approaches: splice regulation
Many splice-modifying Morpholino are targeted to the intronic side of splice junctions in order to block snRNP binding. There are other useful targets for splice-modification though; The DMD drug eteplirsen targets within an exon to block the binding site of an exonic splice enhancer, flipping a regulatory switch that causes exclusion of exon 51 from the mature dystrophin mRNA. The binding sites for splice regulatory proteins, such as exonic splice enhancers, intronic splice suppressors, etc. are also good Morpholino targets but can be more difficult to find; so far, most researchers have used splice junction oligos with a few researchers and some pharmaceutical development companies exploring the splice-regulatory targets.