Open new possibilities in research, therapeutics and beyond with morpholino antisense oligos (2023)

Morpholinos are one of the most elegant and powerful tools in the gene therapy research and development toolbox today. Composed of unique structures that protect against enzymatic degradation, these stable antisense oligonucleotides bind RNA with high specificity to reduce gene expression. In doing so, they offer advantages over other strategies for performing gene knockdown: they target specific gene sequences with minimal off-target effects (unlike other approaches such as siRNA), and they avoid confounding effects of genetic compensation that can obscure the consequences of a deletion. (unlike conventional genetic deletion approaches). These advantages position morpholinos as a key tool in a variety of research and medical applications: they offer researchers a direct means of identifying the consequences of single gene product non-function in fundamental biological research or validation of targets as well as possibilities as therapeutic molecules for a variety of therapeutic areas. Here, we review the unique properties of Morpholinos, their history, and their evolutionary promise.

Not just another oligonucleotide

Antisense oligonucleotides (ASOs) inhibit protein production by binding to RNA molecules. These synthetic single-stranded nucleic acid analogs containing all four nucleotide bases—adenine (A), cytosine (C), guanine (G), and thymine (T)—influence protein synthesis by binding via Watson-base pairing. Crick to RNA species.

Some ASOs achieve gene silencing through induction of RNase H endonuclease activity, resulting in cleavage of the target RNA-DNA heteroduplex via hydrolytic degradation of the RNA. Another class of oligos, steric blocking oligos, are often used to reduce gene expression by binding to the mRNA translation initiation codon and blocking assembly of the mature ribosome. Steric-blocking ASOs can also interfere with the poly-A tail, disrupt the exon splicing process, or interfere with miRNA activity.

Morpholino oligos are a type of sterically blocking ASO. By binding to mRNA, they prevent other macromolecules from interacting with the RNA, but RNase H does not degrade them. Other examples of this class of ASO include locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and 2'-O-methoxyethyl or 2'-O-methoxyphosphorothioates.

Like PNAs, Morpholinos do not have 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 one to explore the specific benefits of uncharged oligos, such as the lack of protein-to-protein interactions that can lead to toxic effects.

Furthermore, morpholinos can be engineered with sufficiently high heat denaturation temperatures (T_metros) to ensure that the balance between bound and unbound forms is directed towards the fully bound side of the equation. The RNA molecule undergoes exonucleolytic activity until finally only an RNA fingerprint remains in the original Morpholino ASO; eventually, this imprint slowly degrades, releasing the single-stranded Morpholino.

Long-term stability of Morpholinos in living systems has been observed in mice.¹Fourteen weeks after Morpholino injection into mouse leg muscle, splice blocking activity was detected by RT-PCR. It has been proposed that morpholinos, when released due to RNA degradation, are free to bind to new RNA targets. Therefore, the morpholino activity decay curve 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 enzyme systems, including nucleases and proteases.²In a separate study, cell-penetrating peptide-conjugated morpholinos were recovered intact from tissues, whereas the peptide was completely degraded.^3,4

From discredited to proven technology

ASOs were proposed by Jim Summerton while doing graduate work at the University of Arizona in Tucson. His first article was rejected by theJournal of Theoretical Biology, with one reviewer referring to the concept as a "dream". Summerton demonstrated the antisense principle while completing his postdoctoral studies at the University of California, Berkeley. He successfully resubmitted a review of his work to theJournal of Theoretical Biology, obtaining 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 brought on board and work soon began with funding from a grant provided by the National Institutes of Health and some investors. By the late 1980s, other ASO companies had entered the field, including Gilead, Genta, Hybridon, Isis, and Triplex.

Morpholinos appeared on the scene in the mid-1980s when Summerton sought to find a substitute for a sugar moiety to alter the properties of ASOs. Using plastic molecular models, he identified that the morpholine ring had the right structure to allow good RNA binding. After trying various ligands, he settled on phosphorodiamidate chemistry, and phosphorodiamidate morpholinos were synthesized in 1989.—this is the backbone of oligo still in use today.

antivirals inc. then it began exploring disease applications for Morpholino ASO, testing its performance against the hepatitis virus, for example. However, pure morpholinos, which remain a core product of Gene Tools, do not readily cross plasma membranes, precluding their use as a treatment for many indications. At AVI Biopharma - a company renamed Antivirals Inc. - it was learned from collaborators that in models of Duchenne muscular dystrophy (DMD), morpholinos were able to enter muscle cells. AVI became Sarepta Therapeutics, which now has three FDA-approved Morpholinos for the treatment of DMD. A group at Children's National Hospital showed that it was actually satellite cells around muscle cells that took up morpholinos. The DMD patients were constantly regenerating their myofibrils. In those treated with Morpholinos, the myofibrils constantly fused with satellite cells that released Morpholinos into the myofibrils.⁶

Other therapeutic applications will require the development of solutions to enhance the cytosolic delivery of Morpholinos. AVI Biopharma has worked on conjugates of morpholinos with cell-penetrating arginine-rich peptides that can enter cells more efficiently, but unfortunately early conjugates affected both diseased and healthy cells, resulting in some concomitant toxicity. That work with cell-penetrating peptides and Morpholinos continues at Oregon State University, with a focus on treatments for influenza, COVID-19 and other viral targets, as well as antibacterial and genetic disease therapies. Sarepta Therapeutics has a peptide-linked morpholino inclinical trials for the treatment of DMD.

Synthesis

Synthesis of morpholino ASOs occurs in a manner analogous to that used for other types of oligonucleotides (eg, by solid-phase synthesis on a resin using an automated synthesizer). The chemistry is different, however. Morpholinos are grown on resin as normal oligo synthesis, but with extension at the 3' end. Compare this to the extension at the 5' end, which is typical in mostex-aliveOligonucleotide synthesis.⁷

A morpholine conversion is also required. The process begins with an RNA nucleoside. The ring of this molecule is oxidized and then reduced in the presence of ammonia to generate the new morpholine ring. The base groups within the molecule are then protected. The subunits are then activated, purified and frozen, then dissolved and loaded onto the synthesizer when needed. Today there are reliable sources of some high purity early subunits, making synthesis much simpler. Gene Tools has plans to explore alternate bases in the future to unlock new possibilities.

Silence Without Noise - A Clean Way To Get Takedowns

The most widespread application of Morpholinos is to achieve genetic drops in the R&D environment. They are mainly used in model organisms, including zebrafish,Xenopus, sea urchins, chickens, and mice, injection into single-celled zygotes being more common, as this approach avoids potential administration problems. In chicks, injection of Morpholinos into the neural tube followed by electroporation is common. Conjugating to a peptide-like moiety that penetrates cells but is organized as a dendrimer, Gene Tools' Vivo-Morpholinos can enter the tissues of adult mammals as well as late stages.Xenopusand zebra fish.

The most common reason to use Morpholinos is to determine what biological mechanisms a specific protein is involved in. Morpholino is injected into an embryo and changes in biological activity are observed to see which pathways are no longer working.

Using Morpholinos to create knockdowns has some clear advantages over more conventional knockout methods, which typically involve mutations or deletions of the gene of interest itself. However, completely deleting or disrupting a gene of interest, from the earliest stages of development, may not lead to an unequivocal phenotype that represents the consequence of the knockout or knockdown of that gene itself, because to the phenomenon of genetic compensation. Many other genes that are closely related and have high sequence similarity to a target gene or that encode proteins with functional similarity to the target gene product can be upregulated when the target gene is null-mutated. Alternatively, the activity of pathways that operate in opposition to the protein encoded by the target gene may be reduced to compensate for the absence of the protein from that gene. Consequently, the phenotype observed in a genetic knockdown may not accurately illustrate the consequence of the absence of the gene product, particularly in more complex organisms with strong compensation potential.

However, a Morpholino provides a more discrete insight into sharp knockdown, which happens when the protein encoded by the target gene is lost without any other change, and provides a powerful complementary technique. Furthermore, selection for the specificity of a Morpholino can be achieved by adding a Morpholino that would normally knock out a given gene into a null mutant with a knockout of that same gene. If no other changes are observed, the specificity of Morpholino is confirmed. This Morpholino can then be inserted into a wild type, and any more extreme phenotypes observed can be attributed to the sharp drop phenotype without the compensated background.

Advantages of specificity

Compared to many other ASOs, Morpholinos offers some advantages in terms of specificity. With siRNA, a translation-suppressed microRNA effect can occur with complementarity to all seven or eight bases of the seed sequence. In fact, it has been shown that knocking out a single target with an siRNA can alter the expression of hundreds of off-target genes.⁸Morpholinos avoid these off-target effects because it takes about 15 bases of complementarity to see good knockdown with a morpholino, minimizing interactions with non-target mRNAs.

This specificity is finally attracting the interest of several companies, as the advantages of using Morpholinos over traditional genetic methods are increasingly appreciated. In fact, a growing number of companies are turning to Gene Tools to access Morpholinos that can be used in preclinical studies.

In therapeutic applications, specificity is also very important. Morpholinos can be easily found that only knock out a specific gene. Also, they do not interact with proteins; they just block the RNA, leading to a very neat mechanism for achieving knockdowns.

Address translation and modification

Com siRNAs y RNase-H⁺oligos that target degradation, there are not many limitations on the target sites. Even mid-exon is suitable, because the process involves cutting the RNA. However, with steric blocking oligos like Morpholinos, there are limitations; some sites work fine, while others don't. Therefore, the location of the link is just as important as the appearance of the link.

Morpholinos can be used to target splicing or translation. Targeting splicing usually requires targeting small nuclear ribonucleoprotein (snRNP) binding sites in introns next to exons and even a little bit on exons. The key is to target the introns right at the margins. Preventing snRNP binding will almost always lead to some activity and can lead to many different outcomes, most of which are likely to be predictable. The binding sites of splicing regulatory proteins can also be blocked.

Targeted translation requires reaching the start codon or moving upstream to the 5' untranslated regions (UTR). Hitting or very close to the start codon is a safe bet, but in many cases targeting a sequence a short distance upstream will achieve the desired effect. It is possible that there is an internal ribosome entry site in the transcript of interest, which could short-circuit the oligo and translate it downstream, but such cases are quite rare in mammalian genomes.

Correct sites can be identified by first performing a BLAST search and verifying the results obtained. However, this type of study will not provide any indication of the importance of binding sites with respect to altering gene expression. Therefore, in order to determine if the identified locations match locations where a Morpholino would likely have an effect, additional excavation is required.

Ultimately, a physical specificity control study should be completed. For example, if two Morpholinos target the same RNA but at slightly different locations and produce the same phenotypic result, the chance that the phenotype is associated with the target and not another gene is much higher. A similar conclusion can be reached for splicing modification if two leading exons are deleted, each causing a frameshift and the same phenotype. If dose synergy is observed when a pair of oligos are administered together, this further supports the idea that the outcome is associated with expected RNA binding. Finally, as discussed above, targeting a null mutant with a Morpholino to the mutant gene is a good assessment of specificity.

delivery improvements

One of the challenges that morpholinos face with respect to their use as therapeutics is their poorlivedelivery into the cytosol of cells. There is a lot of work going on in this area, and progress is being made in terms of more efficient administration and targeting of certain tissues.

Gene Tools Vivo-Morpholinos are easily made morpholinos with a dendrimer at the 3' end of the oligo terminating in guanidinium moieties, similar to the arginine side chains. Each guanidinium is believed to interact with phosphates through both hydrogen bonding and ionic electrostatic interactions, which combined make it a strong cell membrane binder. It can also increase delivery activity by distorting the membrane. Other researchers are exploring the impact of conjugation delivery of many different types of molecules with Morpholinos.

Many groups are also interested in targeting for specific tissues, and various researchers are exploring different approaches. In addition to conjugating specific molecules to morpholinos, others are exploring various physical methods to stimulate targeted delivery of morpholinos, such as radiation to allow entry into tumors and osmotic shock to allow morpholinos to cross the blood-brain barrier.

New technologies creating new opportunities

The emergence of new gene editing tools like CRISPR-Cas9 has created even more opportunities to use morpholinos in basic R&D as well as drug development. For example, nucleic acid switches can be designed that are activated by Morpholinos. For example, intron-exon segments that can be spliced ​​by the addition of a morpholino can be modified in DNA, allowing otherwise non-functional partial proteins to be inserted into the frame by a morpholino splice modifier. . In a therapeutic application, production of a modified endogenous protein at specific times and in specific amounts could be achieved by adjusting the dose and timing of Morpholino treatments.⁹Of course, the current regulatory framework is unlikely to allow such a therapy, but this area is fascinating and worth exploring in animal models.

Meanwhile, Morpholino's therapies have already received some approvals and, with better delivery systems, have the potential to directly affect many disease processes in humans. So while it's tempting to think of crosses with CRISPR and other gene-editing tools, from a regulatory perspective it might be more practical to think of Morpholinos as stand-alone therapies, at least for the next few decades.

Several potential therapeutic targets

Antiviral treatments remain a primary 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 example, in which morpholinos have been shown to decrease the rate of transcription or replication in infected animals.¹⁰Other researchers are studying Morpholinos to treat the flu,¹¹japanese encephalitis,¹²dengue,¹³and the Zika virus.¹⁴

AVI Biopharma previously worked on a Morpholino engineered against the Ebola virus genome in response to a potential infection by a USAMRIID (United States Army Medical Research Institute of Infectious Diseases) technician.^15-17The drug was not necessary, but the rapid response illustrated how quickly morpholinos can adapt to a new viral threat. The development of Morpholinos as rapid antivirals against bioterrorist or hostile military viruses represents an exciting opportunity.

Non-Pharmaceutical Applications for Morpholinos

Morpholinos have attracted interest outside of the pharmaceutical industry as potential tools for various applications. Perhaps most notable is its use to sterilize farmed fish. Genetically modified fish represent a significant opportunity, but their release into the environment is highly frowned upon. Sterilization ensures that even if the fish escape into the wild, they will not affect the natural gene pool. Morpholinos can be used to delete a gene within a narrow time frame during early development that prevents gametes from developing.¹⁸

Handling Agent Toxicity

Pure morpholinos pose minimal safety concerns, largely due to their low delivery; the majority is excreted in the urine. With effective delivery moieties bound to Morpholinos (eg, Live Morpholinos or cell-penetrating peptides), significant delivery can be achieved.

With this increased delivery comes the potential for concomitant toxicity. Live Morpholinos, cell penetrating peptides and other delivery agents may have some level of toxicity. Careful design of the delivery agent is essential to maintain a balance between efficacy and toxicity. The toxicity of the oligo sequence itself should also be assessed, with toxicity due to elimination of its intended target or interaction with unexpected RNA (morpholino specificity is good but not perfect).

Genetic tools: advancing the promise of morpholinos

The core mission ofgenetic toolsis the production of oligonucleotides, specifically Morpholinos, in a clean, fast and reliable way. The goal is to support all companies and academic laboratories that need Morpholinos for use in basic research or for preclinical work. While big pharmaceutical companies, particularly those with a more molecular bent, have an interest in morpholinos as a valuable tool, most of the activity in the industry comes from new and emerging companies that believe morpholinos are an important tool for accelerate the launch of their candidates to the market, sometimes as a pharmaceutically active ingredient.

Gene Tools currently only manufactures Morpholinos for R&D applications. Large-scale production of GMP material is being considered, but would require significant investment. There are some other companies that provide GMP manufacturing but do not support research scale like Gene Tools. As such, we have established a niche position within a larger ecosystem.

However, the company is in the process of spinning off from a company focused on therapeutic applications.

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