February 1, 2023 PAO-01-23-CL-05
Antisense oligonucleotides (ASOs) inhibit protein production by binding to RNA molecules. These synthetic short, single-stranded nucleic acid analogs containing the four nucleotide bases — adenine (A), cytosine (C), guanine (G), and thymine (T) — influence protein synthesis by binding via Watson–Crick base pairing to specific RNA species.
Some ASOs achieve gene silencing through induction of the endonuclease activity of RNase H, which results in the cleavage of the target RNA–DNA heteroduplex by hydrolytic RNA degradation. Another class of oligos, the steric blocking oligos, are often used to knock down gene expression by binding to the translation initiation codon of mRNA and blocking assembly of the mature ribosome. Steric blocking ASOs can also interfere with poly-A tailing, alter the exon splicing process, or interfere with miRNA activity.
Morpholino oligos are a type of steric-blocking ASO. By binding to mRNA, they prevent other macromolecules from interacting with the RNA, but are not degraded by RNase H. Other examples of this class of ASO include locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and 2'-O-methoxy ethyl or 2 '-O-methoxy phosphorothioates.
Like PNAs, Morpholinos do not carry a net charge. Unlike PNAs, however, they still exhibit fairly decent water solubility, which is a crucial attribute. The combination of neutrality and water solubility allows exploitation of the benefits specific to uncharged oligos, such as a lack of interactions with proteins that can lead to toxic effects.
In addition, Morpholinos can be designed with sufficiently high thermal denaturation temperatures (Tms) to ensure that the equilibrium between the bound and unbound forms is driven toward the fully bound side of the equation. The RNA molecule is subjected to exonucleolytic activity until eventually only an RNA footprint on the original Morpholino ASO remains; eventually, this footprint slowly degrades away and releases the single-stranded Morpholino.
The long-term stability of Morpholinos in living systems has been observed in mice.1 Fourteen weeks after injection of a Morpholino into the leg muscle of a mouse, splice-blocking activity was detected using RT-PCR. It has been proposed that the Morpholinos, when released due to RNA degradation, are free to bind to new RNA targets. Thus, the decay curve for Morpholino activity is asymptotic, with the asymptotic level of activity determined by the rate of RNA degradation and oligo release. Morpholinos have also been shown to be stable when exposed to many different enzymatic systems, including nucleases and proteases.2 In a separate study, Morpholinos conjugated to cell-penetrating peptides were recovered intact from tissues, while the peptide was fully degraded.3,4
ASOs were proposed by Jim Summerton while pursuing his graduate degree at the University of Arizona in Tucson. His first paper was rejected by the Journal of Theoretical Biology, with one reviewer referring to the concept as a “pipe dream.” Summerton proceeded to prove the principle of antisense while completing postdoctoral studies at the University of California, Berkeley. He successfully resubmitted a revision of his work to the Journal of Theoretical Biology, getting the original submission date included in the resubmitted manuscript.5
In 1980, Summerton left academia to develop and commercialize his patentable ideas. Antivirals Inc. was incorporated, and work started soon with funding from a grant provided by the National Institutes of Health and a few investors. By the late 1980s, other ASO companies had entered the field, including Gilead, Genta, Hybridon, Isis, and Triplex.
Morpholinos entered the picture in the mid-1980s when Summerton sought to find a replacement for a sugar moiety to change the properties of ASOs. Using plastic molecular models, he identified the morpholine ring as having the right structure to allow good binding to RNA. After trying various linkers, he settled on phosphorodiamidate chemistry, and phosphorodiamidate Morpholinos were synthesized in 1989 — this is the oligo backbone still in use today.
Antivirals Inc. then began exploring disease applications for Morpholino ASOs, testing their performance against hepatitis virus, for instance. However, bare Morpholinos, which remain a primary product for Gene Tools, do not easily cross plasma membranes, which preclude their use as a therapeutic for many indications. At AVI Biopharma — a renamed Antivirals Inc. — it was learned with collaborators that, in Duchenne muscular dystrophy (DMD) models, Morpholinos were able to enter muscle cells. AVI went on to become Sarepta Therapeutics, which now has three Morpholinos FDA-approved for the treatment of DMD. A group at Children’s National Hospital then showed that it was actually satellite cells around the muscle cells that were taking up the Morpholinos. The DMD patients were undergoing constant regeneration of their myofibrils. In those treated with Morpholinos, the myofibrils were thus constantly fusing with satellite cells that were releasing Morpholinos into the myofibrils. 6
Further therapeutic applications will require the development of solutions to improve the cytosolic delivery of Morpholinos. AVI Biopharma had worked on conjugates of Morpholinos with cell-penetrating, arginine-rich peptides that could enter cells more effectively, but unfortunately the early conjugates impacted both diseased and healthy cells, resulting in some attendant toxicity. That work with cell-penetrating peptides and Morpholinos is continuing at Oregon State University, with a focus on treatments of influenza, COVID-19, and other viral targets, as well as antibacterials and genetic disease therapies. Sarepta Therapeutics has a peptide-linked Morpholino in clinical trials for treatment of DMD.
Synthesis of Morpholino ASOs proceeds in a manner analogous to that used for other types of oligonucleotides (e.g., via solid-phase synthesis on a resin using an automated synthesizer). The chemistry is different, however. The Morpholinos are grown on resin like normal oligo synthesis, but with extension of the 3'-end. Contrast this with extension at the 5'-end, which is typical in most ex vivo syntheses of oligonucleotides.7
A morpholine conversion is also required. The process starts with an RNA nucleoside. The ring of this molecule is oxidized open and then reduced in the presence of ammonia to generate the new morpholine ring. The base groups within the molecule are then protected. Next, the subunits are activated, purified, and frozen, then dissolved and loaded onto the synthesizer when needed. Today, there are reliable sources of some high-purity starting subunits, which makes the synthesis much simpler. Gene Tools has plans to explore alternative bases in the future to unlock new possibilities.
The most widespread application for Morpholinos is to achieve genetic knockdowns in the R&D setting. They are used mostly in model organisms — including zebrafish, Xenopus, sea urchins, chicks, and mice — with injection into single-cell zygotes most common, as this approach avoids potential delivery problems. In chicks, it is common to inject Morpholinos at the neural tube, followed by electroporation. Vivo-Morpholinos from Gene Tools, which are conjugated to a moiety similar to a cell-penetrating peptide but arranged as a dendrimer, can enter the tissues of adult mammals, as well as later-stage Xenopus and zebrafish.
The most common reason for using Morpholinos is to determine in which biological mechanisms a specific protein is involved. The Morpholino is injected into an embryo, and changes in biological activity are observed to see which pathways are no longer functioning.
Using Morpholinos to create knockdowns presents some clear advantages over more conventional knockout methods, which often involve mutations or deletions of the gene of interest itself. However, completely deleting or disrupting a gene of interest, from the earliest phases of development, may not lead to an unambiguous phenotype depicting the consequence of the absence (knockout) or reduction (knockdown) of that gene on its own, owing to the phenomenon of genetic compensation. Many other genes that are closely related and have great sequence similarity to a targeted gene or that encode proteins with functional similarity to the product of the targeted gene may be upregulated when the target gene is mutated to null. Alternatively, the activity of pathways that operate in opposition to the protein encoded by the targeted gene may be reduced to compensate for the absence of that gene’s protein. Consequently, the phenotype observed in a genetic knockdown may not accurately illustrate the consequence of the gene product’s absence, particularly in more complex organisms with robust compensation potential.
A Morpholino, however, provides a more discrete view of the acute knockdown — what happens when the protein encoded by the target gene is lost without any other changes — and provides a powerful complementary technique. In addition, screening of the specificity of a Morpholino can be achieved by adding a Morpholino that would normally knock down a given gene into a null mutant with a genetic knockout of that same gene. If no other changes are observed, the specificity of the Morpholino is confirmed. That Morpholino can then be inserted into a wild type, and any more extreme phenotype that is observed can be attributed to the phenotype of the acute knockdown without the compensated background.
Compared with many other ASOs, Morpholinos offer some advantages in terms of specificity. With siRNA, a microRNA effect with suppression of translation can occur with complementarity of the seven or eight bases of the seed sequence. In fact, it has been shown that knocking down a single target with an siRNA could change the expression of hundreds of off-target genes.8 Morpholinos avoid those off-target effects because it takes about 15 bases of complementarity to observe good knockdown with a Morpholino, minimizing interactions with mRNAs other than the target.
This specificity is finally attracting the interest of a range of companies as the advantages of using Morpholinos rather than traditional genetic methods are becoming more widely appreciated. Indeed, increasing numbers of companies are coming to Gene Tools for access to Morpholinos that can be used in preclinical studies.
In therapeutic applications, specificity is very important as well. Morpholinos can easily be found that only knock down a specific gene. In addition, they do not interact with proteins; they only block RNA, leading to a very clean mechanism for achieving knockdowns.
With siRNAs and RNase-H+ oligos that target degradation, there are not many limitations on target sites. Even the middle of the exon is suitable, because the process involves cutting the RNA. With steric-blocking oligos like Morpholinos, however, there are limitations; some sites work well, while others do not. Therefore, the location of binding is as important as the occurrence of binding.
Morpholinos can be used to target splicing or translation. Targeting splicing usually requires hitting small nuclear ribonucleoprotein (snRNP) binding sites in the introns next to the exons, and even a little bit into the exons. The key is targeting the introns right at the margins. Prevention of snRNP binding will almost always lead to some activity, and it can lead to many different outcomes, with most of the likely ones predictable. Splice regulatory protein binding sites can also be blocked.
Targeting translation requires hitting the start codon or moving upstream into the 5’-untranslated regions (UTRs). Hitting the start codon or very close to it is a safe bet, but, in many cases, targeting a sequence a short distance upstream will achieve the desired effect. It is possible for an internal ribosome entry site to be in the transcript of interest, which can short-circuit the oligo and translate downstream from it, but such instances are pretty rare in mammalian genomes.
The right sites can be identified by first conducting a BLAST search and checking the results obtained. However, this type of study will not provide any indication of the significance of the binding sites with respect to altering gene expression. Thus, to determine whether the identified sites match sites where a Morpholino would be likely to have an effect, further digging is required.
Ultimately, a physical specificity control study must be completed. For instance, if two Morpholinos target the same RNA but at slightly different sites and produce the same phenotypic outcome, the likelihood is much greater that the phenotype is associated with hitting the target and not with hitting another gene. A similar conclusion can be made for splice modification if two early exons are taken out that each cause a frameshift and the same phenotype. If dose synergy is observed when such a pair of oligos is administered together, it further supports the idea that the outcome is associated with binding the expected RNA. Finally, as discussed before, targeting a null mutant with a Morpholino for the mutant gene is a good specificity assessment.
One of the challenges that Morpholinos face with respect to their use as therapeutics is their poor in vivo delivery into the cytosol of cells. Much work is being done in this area, with advances being made with respect to improved efficiency of delivery and targeting of certain tissues.
Vivo-Morpholinos from Gene Tools are readily manufacturable Morpholinos with a dendrimer on the 3′ end of the oligo terminating in guanidinium moieties, similar to the side chains of arginines. Each guanidinium is thought to interact with phosphates through two hydrogen bonds and ionic electrostatic interactions, which combined make it a tight cell membrane binder. It may also increase delivery activity by distorting the membrane. Other researchers are exploring the impact on delivery of conjugating many different types of molecules to Morpholinos.
Many groups are also interested in tissue-specific targeting, and various researchers are exploring different approaches. In addition to conjugating specific molecules to Morpholinos, others are exploring various physical methods to encourage the targeted delivery of Morpholinos, such as radiation to enable entry into tumors and osmotic shock to enable Morpholinos to cross the blood–brain barrier.
The emergence of new gene-editing tools, such as CRISPR-Cas9, has created even more opportunities for the use of Morpholinos in basic R&D, as well as in drug development. For instance, nucleic acid switches can be designed that are activated by Morpholinos. As an example, intron–exon segments that can be spliced out by adding a Morpholino can be designed into DNA, allowing partial proteins that would otherwise be nonfunctional to be brought in-frame by a splice-modifying Morpholino. In a therapeutic application, production of an engineered endogenous protein at specific times and in specific quantities could potentially be achieved by adjusting the dose and timing of Morpholino treatments.9 Of course, the current regulatory framework is unlikely to allow such a therapy, but this area is a fascinating one worth exploring in animal models.
In the meantime, Morpholino therapeutics have already seen some approvals and with better delivery systems have the potential to directly affect a lot of disease processes in humans. Therefore, while it is tempting to think about crossovers with CRISPR and other gene-editing tools, from a regulatory standpoint it may be more practical to think about Morpholinos as standalone therapies, at least for the next few decades.
Antiviral treatments remain a key focus for companies exploring the therapeutic potential of Morpholinos. One target is the SARS-CoV-2 virus that causes COVID-19. Preclinical work is ongoing, for instance, in which Morpholinos have been shown to slow down the rate of transcription or replication in infected animals.10 Other researchers are looking at Morpholinos to treat influenza,11 Japanese encephalitis,12 dengue,13 and Zika virus.14
AVI Biopharma previously worked on a Morpholino designed against the Ebola virus genome in response to a potential infection of a USAMRIID (United States Army Medical Research Institute of Infectious Diseases) technician.15–17 The drug wasn’t needed, but the rapid response illustrated how quickly Morpholinos can be adapted to a new viral threat. The development of Morpholinos as rapid antivirals against bioterror or hostile military viruses represents an interesting opportunity.
Bare Morpholinos present minimal safety concerns, largely due to their poor delivery; most is excreted in urine. With effective delivery moieties attached to Morpholinos (e.g., Vivo Morpholinos or cell-penetrating peptides), significant delivery can be achieved.
With that higher delivery comes the potential for attendant toxicity. Vivo Morpholinos, cell-penetrating peptides, and other delivery agents can have a certain level of toxicity. Careful design of the delivery agent is essential to maintain a balance between efficacy and toxicity. Toxicity of the oligo sequence itself must also be assessed, with toxicity either due to knockdown of its intended target or interaction with unexpected RNA (Morpholino specificity is good but not perfect).
The core mission of Gene Tools is the production of oligonucleotides, specifically Morpholinos, cleanly, rapidly, and reliably. The goal is to support all companies and academic labs that need Morpholinos for use in basic research or for preclinical work. While Big Pharma companies, particularly those with a more molecular bent, have interest in Morpholinos as a valuable tool, the greatest activity in industry comes from startups and emerging companies that believe that Morpholinos are an important tool for accelerating their candidates to the market, sometimes as the pharmaceutically active ingredient.
Currently, Gene Tools only makes Morpholinos for R&D applications. The production of GMP material at large scale is under consideration but would require significant investment. There are a few other companies providing GMP manufacturing, but that do not offer research-scale support like Gene Tools. As such, we have established a niche position within a bigger ecosystem.
The company is, however, in the process of spinning off a company focused on therapeutic applications.
Jon Moulton is a scientist with Gene Tools LLC. He covers many functions with ever changing tasks, officially called the Biology and Special Projects guy. He designs antisense sequences, builds equipment, exhibits at conferences, writes, helps with experimental design, fixes things, maintains a literature database, and troubleshoots. Jon holds degrees in chemistry and biology and a Ph.D. in environmental sciences and resources: biology from Portland State University. He was hired at Gene Tools in 1999, when there were four employees.