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

Table of Contents
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 From discredited to proven technology Synthesis Silence Without Noise - A Clean Way To Get Takedowns Advantages of specificity Address translation and modification delivery improvements New technologies creating new opportunities Several potential therapeutic targets Handling Agent Toxicity Genetic tools: advancing the promise of morpholinos FAQs Videos

February 1, 2023PAO-01-23-CL-05

    • Label:
    • genetics
    • ARN
    • Oligonucleotides
    • Therapeutic Sarepta
    • Antisense Oligonucleotides
    • Q12023
    • genetic tools
    • morpholinos
    • therapeutic oligos
    • exon skipping

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.

(Video) What are oligonucleotide therapeutics, and how do they work?

Furthermore, morpholinos can be engineered with sufficiently high heat denaturation temperatures (Tmetros) 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 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

(Video) A Visual Exploration of Antisense Oligonucleotide Therapies in ALS

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.

(Video) Antisense Oligonucleotides- Mechanisms of Action and Rational Design

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.

(Video) Antisense Oligonucleotide Therapy for ALS

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.

(Video) Annemieke Aartsma-Rus: Design of Exon Skipping Oligonucleotides

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


  1. dj of pitsGene doping: the hype and the reality.”J. Pharmacol.154: 623–631 (2008).
  2. Hudziak R.M. and anotherResistance of morpholino phosphorodiamidate oligomers to enzymatic degradation”. Antisense Nucleic Acid Drug. Dev.6: 267–272 (1996).
  3. Schissel, Carly K. et al.Cell-penetrating peptides retain antisense morpholino oligomer delivery activity.”ACS Bio. HOW. chemistry2: 150–160 (2022).
  4. Youngblood DS et al. Stability of morpholino oligomer cell penetrating peptide conjugates in human serum and in cells.chemical bioconjugator18: 50–60 (2007).
  5. SUMMERTON, James.Intracellular inactivation of specific nucleotide sequences: a general approach for the treatment of viral diseases and virus-mediated cancers”.J.Teor. Biol.78: 61–75 (1979; first presentation 1973).
  6. Novak, James S. et al.Myoblasts and macrophages are required for therapeutic delivery of morpholino antisense oligonucleotides to dystrophic muscle..”Nature Communications.8: 941 (2017).
  7. Summerton, James y D. Weller.Morpholino antisense oligomers: design, preparation and properties.”Development of antisense nucleic drugs.7: 187–195 (1997).
  8. SCACHERI, P. C. et al.Short interfering RNAs can induce unexpected and divergent changes in non-targeting protein levels in mammalian cells." PNAS.101: 1892–1897 (2004).
  9. Cripe T.P. and anotherHarnessing gene therapy to achieve continuous or long-term controllable expression of biotherapeutics.”Science Adv.8: eabm1890 (2022).
  10. Laghi, Valerio et al.Exploration of zebrafish larvae as a model of COVID-19: probable abortive replication of SARS-CoV-2 in the swim bladder.”Forehead. Cell. Infect. Microbiol.12 (2022).
  11. Böttcher-Friebertshäuser E. et al.Inhibition of Influenza Virus Infection in Cultured Human Airway Cells by a Morpholino Oligomer Conjugated to an Antisense Peptide Targeting the TMPRSS2 Hemagglutinin-Activating Protease.”J.Virol.85: 1554–62 (2011).
  12. Nazmi, Arshed, Kallol Dutta and Anirban Basu.Antiviral and neuroprotective role of morpholino oligomers conjugated with octaguanidine dendrimers in Japanese encephalitis.PLoS neg. Too much. Say.4: e892 (2010).
  13. Phumesin, Patta et al.Vivo-morpholino oligomers strongly inhibit dengue virus replication and production.”Bow. Virol.163: 867–876 (2018).
  14. Popik, Waldemar et al.Morpholino phosphorodiamidate targeting the 5' untranslated region of ZIKV RNA inhibits virus replication.”Virology.519: 77–85 (2018).
  15. Enterlein, Sven et al.VP35 Knockdown Inhibits Ebola Virus Amplification and Protects Against Lethal Infection in Mice.”antimicrobial Chemotherapy agents. 50: 984–993 (2006).
  16. Vander-Linden CLEbola-specific gene therapies protect non-human primates from deadly disease.”Trimestral Chem-Bio Defense.3: 8–9 (2006).
  17. Kortepeter, MG et al.Management of potential laboratory exposure to Ebola virus using a patient biocontainment treatment unit.emerge infect Dis.14: 881–887 (2008).
  18. Wong T. T. y Y. Zohar.Production of reproductively sterile fish using a non-transgenic gene silencing technology.”Scientific Representative5:15822. (2015)

    • Label:
    • genetics
    • ARN
    • Oligonucleotides
    • Therapeutic Sarepta
    • Antisense Oligonucleotides
    • Q12023
    • genetic tools
    • morpholinos
    • therapeutic oligos
    • exon skipping


What is an antisense morpholino? ›

A Brief Introduction to Morpholino Antisense. Morpholino oligos are advanced tools for blocking sites on RNA to obstruct cellular processes. A Morpholino oligo specifically binds to its selected target site to block access of cell components to that target site.

What are morpholino antisense oligonucleotides? ›

Morpholinos are synthetic antisense oligonucleotides (around 25 nucleotides) designed to bind and block the translation initiation complex of messenger RNA (mRNA) sequences. This technology has been used to test the role of specific genes by transient blocking, particularly during development.

What is the purpose of Morpholino? ›

Morpholinos are typically used to block translation of mRNA and to block splicing of pre‐mRNA, though they can block other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non‐antisense effects.

What are the disadvantages of antisense oligonucleotides? ›

Disadvantages include increasing the complexity of the formulation and the potential to induce toxicity separate from the active dsRNA. Nanoparticles can alter the pharmacokinetics and the biodistribution of oligonucleotides and these new properties should be characterized with care.

What is antisense therapy used for? ›

Antisense oligonucleotide therapy has also been used to treat spinal muscular atrophy type 1 (SMA1), an autosomal recessive motor neuron degenerative disorder that affects approximately 1 in 10,000 newborns.

How long does Morpholino last? ›

Morpholinos, as chemically modified oligonucleotides, effectively bind target mRNA and block translation thereby knocking down expression. Embryos are injected at the one-cell to four-cell stage, and efficacy typically lasts for 5 days but may vary with dose.

Which Morpholino is FDA approved? ›

The US Food and Drug Administration today (19 Sep 2016) granted Accelerated Approval to eteplirsen (EXONDYS 51), a Morpholino oligo-based treatment for some forms of Duchenne muscular dystrophy (DMD). This is the first approval of a Morpholino drug.

How does antisense oligonucleotide therapy work? ›

ASOs offer new opportunities for therapeutic intervention because they act inside the cell to influence protein production. Once inside the cell, the ASO binds to the target mRNA or pre-mRNA, inducing its degradation and preventing the mRNA from being translated into a detrimental protein product.

How can antisense oligonucleotides be used as a therapeutic agent? ›

Antisense therapy has been useful in the treatment of cardiovascular disorders such as restenosis after angioplasty, vascular bypass graft occlusion, and transplant coronary vasculopathy. Antisense oligonucleotides also have shown promise as antiviral agents.

What are the advantages of Morpholino? ›

There is no other gene knockdown reagent (including siRNA, PNA, mPNA, S-DNA, and LNA) that combines the properties of stability, nuclease-resistance, efficacy, long-term activity, water-solubility, low toxicity and exquisite specificity. Only Morpholino oligos provide all of these.

What is the mechanism of action of Morpholino? ›

Morpholinos do not trigger the degradation of their target RNA molecules, unlike many antisense structural types (e.g., phosphorothioates, siRNA). Instead, Morpholinos act by "steric blocking", binding to a target sequence within an RNA, inhibiting molecules that might otherwise interact with the RNA.

How to order Morpholino? ›

Gene Tools Design Request; use our Morpholino design request website and let us design your Morpholinos prior to ordering.
  1. Gene Tools ONLINE STORE; place your order here using a credit card or purchase order.
  2. Fax Order Form (requires Adobe Acrobat 5)
  3. Here is the price list effective 1 July 2022.

Is antisense therapy FDA-approved? ›

Antisense and small interfering RNA (siRNA) oligonucleotide therapeutics have been FDA-approved in recent years to treat rare diseases; in addition, many oligonucleotide therapeutics are currently in development to treat common chronic diseases.”

What are the challenges of antisense technology? ›

Technical problems in antisense technology

These issues included inadequate tissue distribution and cell permeability, as well as low specificity to their mRNA targets. High specificity is necessary to prevent toxicity by inhibition of undesired proteins in fundamental cellular processes.

How many antisense oligonucleotides are FDA-approved? ›

Over 20 years of research and clinical trials, 14 small nucleic acid-based drugs have entered the market by 2021. The FDA-approved oligonucleotide drugs are listed in the table below.

What is the benefit of antisense oligonucleotide? ›

Depending on sequence and modifications, antisense oligonucleotides can alter RNA function through several distinct mechanisms, making them a diverse tool. They can be used to restore protein expression, reduce expression of a toxic protein, or modify mutant proteins to reduce their toxicity.

Which antisense drugs are approved by FDA? ›

The ADME of ASO drugs contributes to their unique set of ADRs and toxicity. In this review, to better understand their ADME, the ten FDA-approved ASO drugs were selected, including Fomivirsen, Pegaptanib, Mipomersen, Nusinersen, Inotersen, Defibrotide, Eteplirsen, Golodirsen, Viltolarsen, and Casimersen.

How do you knock down a gene? ›

In research laboratories, genes have traditionally been knocked down using small interfering RNA (siRNA) or short hairpin RNA (shRNA). These methods are still useful, but newer options using catalytically dead Cas9 (dCas9) or Cas13 proteins are also available. These CRISPR-based methods can offer advantages.

How do you dilute morpholinos? ›

Prepare Morpholino solution and microinjector
  1. Make a dilution from the Morpholino stock solution in a microcentrifuge tube so that a 1‐ng to 10‐ng dose of Morpholino can be delivered in a 2‐nl to 5‐nl injection.
  2. Add 1 volume of 1% aqueous phenol red solution to 9 volumes of Morpholino injection solution (final 0.1%).
Jul 1, 2008

When was morpholinos invented? ›

Morpholino oligos were devised by James Summerton in 1985 and were developed at ANTIVIRALS Inc. (now Sarepta Therapeutics Inc.), the pioneer antisense company founded by Summerton in 1980. James Summerton, Ph. D. is now the Manager of Gene Tools, LLC.

How many oligonucleotide drugs have been approved? ›

Fifteen oligonucleotide therapeutics have been approved for various rare diseases, however, there is still a significant need to develop more drugs in the market.

How many gene therapies are approved by the FDA? ›

As of June 2021, the FDA had approved 2 gene therapy products.

How many synthetic color additives are approved by the FDA? ›

There are nine certified color additives approved for use in the United States (e.g., FD&C Yellow No. 6. See chart for complete list.). Certified food colors generally do not add undesirable flavors to foods.

How long do antisense oligonucleotides work? ›

Depending on the chemistry and chemical design, the effects of single-stranded phosphorothioate ASOs on gene expression can last from 6 weeks to more than 6 months following a single injection, supporting infrequent administration of the antisense drug (Hua et al.

What are the different types of therapeutic oligonucleotides? ›

Classification of oligonucleotide therapeutics. ssDNA/RNA: Single-stranded DNA/RNA, dsRNA: double-stranded RNA, shRNA: small hairpin RNA, sgRNA: single-guide RNA.

Which type of innovation can be achieved using antisense RNA? ›

5. Which type of inhibition can be achieved using antisense RNA? Explanation: Transient inhibition of particular genes can be achieved by directly introducing antisense RNA or antisense oligonucleotides into cells.

What are the uses of synthetic oligonucleotides? ›

Synthetic oligonucleotides (ODNs) are short nucleic acid chains that can act in a sequence specific manner to control gene expression.

Where do morpholinos bind? ›

Morpholinos are nucleotide analogs that recognize and bind short sequences (about 25 nucleotides) at the transcription start site or at splice sites of pre-mRNAs, and thus block the translation or proper splicing of the mRNA (Summerton & Weller, 1997).

Why are morpholinos used in zebrafish? ›

Morpholino oligonucleotides (MOs) are the most widely used anti-sense knockdown tools in the zebrafish (Danio rerio) community. MOs have been used to accelerate gene discovery through large-scale screening,1,2 to probe candidate gene function,3 and to verify mutant phenotypes.

How does oligonucleotide microarray work? ›

Oligonucleotide microarrays is the name used to describe a specific way of manufacturing probes for an array by producing short oligonucleotide sequences which represent a gene. This sequence is synthesized directly onto the array.

What is a PMO oligo? ›

Phosphorodiamidate morpholino oligomers (PMOs) are synthetic DNA analogs that inhibit gene expression in a sequence-dependent manner. PMOs of various lengths (7 to 20 bases) were tested for inhibition of luciferase expression in Escherichia coli. Shorter PMOs generally inhibited luciferase greater than longer PMOs.

What is antisense gene silencing? ›

Antisense mediated gene silencing refers to the post-transcriptional silencing of genes using small sequence specific (anti-sense) molecules that through complementary base pairing suppress translation or direct degradation of specific target mRNAs.

How much do oligonucleotides cost? ›

Primer mix DNA
25 nmole DNA Plate Oligo15 - 60 Bases$0.2200 USD / Base
100 nmole DNA Plate Oligo10 - 90 Bases$0.3600 USD / Base
250 nmole DNA Plate Oligo5 - 100 Bases$0.6000 USD / Base
1 umole DNA Plate Oligo5 - 100 Bases$1.2500 USD / Base

Which type of oligo purification should I choose? ›

We recommend HPLC purification for oligos <10 bases in length and for oligos bearing hydrophobic modifiers that are easily enriched using a reverse-phase approach (see below). Mass recovery usually runs 20-50% for PAGE and 50-70% for HPLC.

What is antisense drug delivery? ›

The antisense molecules are synthetic replica of specific mRNA sequence to block the function of the specific target gene of interest in the human genome. Recently, antisense therapy has emerged as a promising tool to treat various diseases, and for treatment, several antisense drugs have been approved by the FDA.

How much does antisense oligonucleotide therapy cost? ›

One of the key challenges in the delivery of ASOs is the current cost of treatment. The cost of eteplirsen is estimated at US$57,600 per month. For nusinersen, used in the treatment of SMA, costs per injection are US$125,000, first-year treatment costs reach US$750,000, and costs are US$375,000 every year thereafter.

Who invented antisense therapy? ›

Practical application of the principles of antisense oligonucleotides (ASOs) was first formulated in Novosibirsk (Russia) by Grineva in 1967 [1].

Is mRNA made from sense or antisense? ›


In a cell, antisense DNA serves as the template for producing messenger RNA (mRNA), which directs the synthesis of a protein.

What are the advantages of antisense technology? ›

Antisense technology plays an important role in generating high yielding, disease and pest resistant, high nutritional value, and stress-tolerant crop varieties.

What is antisense technology technique? ›

Antisense DNA technology is a method to inhibit or downregulate the production of a target protein by using antisense DNA or RNA molecules. An antisense sequence is a DNA or RNA that is perfectly complementary to the target nucleotide sequence present in the cell.

Is antisense positive or negative? ›

On the basis of genome type, single-stranded RNA viruses can be classified into positive and negative sense RNA viruses. The positive sense RNA virus is also referred to as sense strand or plus-strand, while on the other hand, negative sense RNA is also referred to as antisense or minus strand.

Which nanoparticles are FDA approved? ›

Table 1 List of FDA-approved nanotechnology-based products and clinical trials.
Polymer nanoparticles-synthetic polymer particles combined with drugs or biologics
NameMaterial description
DepoCyt© (Sigma-Tau)Liposomal cytarabine
Marqibo® (Onco TCS)Liposomal vincristine
Onivyde® (Merrimack)Liposomal irinotecan
55 more rows

How many Nanomedicines are approved? ›

Nevertheless, as per the published reports, the US Food and Drug Administration and European Medicines Agency have approved the use of nearly 70 nanomedicines against human diseases since 1995, while twice the number of formulations are now either at an early stage or finishing stage of clinical studies [295] .

How many ASOs are approved? ›

To date, three RNase H-competent ASOs have received regulatory approval; fomivirsen, mipomersen and inotersen (Fig. 1a–c; Table 1). Fig. 2: Oligonucleotide-mediated gene regulatory mechanisms.

What does antisense mean in genetics? ›

Antisense is the non-coding DNA strand of a gene. In a cell, antisense DNA serves as the template for producing messenger RNA (mRNA), which directs the synthesis of a protein.

What is the antisense of DNA? ›

Small pieces of DNA that can bind to specific molecules of RNA and block the cell's ability to use the RNA to make a protein or work in other ways. Antisense DNA may be used to block the production of proteins needed for cell growth. It is being studied in the treatment of many types of cancer.

What is antisense miRNA? ›

Anti-miRNA oligonucleotides (AMOs) represent antisense oligonucleotides (ASOs), single-stranded, chemically modified DNA-like molecules that are 17 to 22 nt in length and designed to be complementary to a selected miRNAs. Thus they are able to specifically inhibit expression of that gene.

What is antisense and sense in DNA? ›

A DNA segment encoding a protein usually has a "sense" strand and a complementary "antisense" strand which acts as a template for RNA polymerase. Conventionally, the sense strand is considered to encode the protein since it has the same sequence as the mRNA.

Is antisense therapy a type of gene therapy? ›

Antisense gene therapy is a gene silencing technique similar to RNA interference, but uses a slightly different mechanism. The therapy is called a gene silencing technique because, instead of repairing the gene, it aims to “silence” the gene's effect.

Why is it called antisense? ›

The second strand is called the antisense strand because its sequence of nucleotides is the complement of message sense. When mRNA forms a duplex with a complementary antisense RNA sequence, translation is blocked.

What is true about antisense technology? ›

Antisense technology is a tool used for gene expression inhibition. The principle associated with antisense technology is that an antisense nucleic acid sequence base pairs with a complementary sense RNA strand. This pairing prevents it from being translated into a protein which is a macromolecule.

What produces antisense RNA? ›

Antisense RNA is synthesized by in-vitro transcription using highly specific DNA-dependent RNA polymerases derived from bacteriophages such as SP6, T7, and T3 polymerases.

How does an antisense gene control gene expression? ›

Antisense transcripts are wide-spread throughout eukaryotic genomes and are generated from independent, bidirectional or cryptic promoters. Antisense transcription/transcripts regulate gene expression and genome integrity via transcriptional interference, histone modification, and/or DNA methylation.

Is antisense oligonucleotide a drug? ›

A drug used to treat certain mutations that cause Duchenne muscular dystrophy (DMD).

What are antisense molecules give examples? ›

The term antisense molecules comprises several classes of oligonucleotide molecules that contain sequence complementarity to target RNA molecules, such as mRNA, viral RNA, or other RNA species, and that inhibit the function of their target RNA after sequence-specific binding.

Is mRNA same as sense or antisense? ›

Strictly speaking, only the mRNA makes "sense" with the genetic code, as the translated protein peptide sequence can be directly inferred from this strand. The "antisense" strand of DNA is complementary to the "sense" strand and is the actual template for mRNA synthesis.

Is RNA sense or antisense? ›

Double-stranded RNA can also act as a catalytic, enzyme-dependent antisense agent through the RNAi/siRNA pathway, involving target mRNA recognition through sense-antisense strand pairing followed by target mRNA degradation by the RNA-induced silencing complex (RISC).


1. Antisense Oligonucleotides for Prion Disease
(CJD Foundation)
2. David Corey: Guidelines for Experiments using Antisense Oligonucleotides and Double-Stranded RNAs
(Oligonucleotide Therapeutics Society)
3. Antisense Oligonucleotides Part 1
(Elliot Nicholson)
4. Chemical and Nanotechnology based Approaches to Build Next gen Oligonucleotide Therapeutics
(Oligonucleotide Therapeutics Society)
5. Art Krieg: How to Test for Immune Activation by your Therapeutic Oligonucleotide
(Oligonucleotide Therapeutics Society)
6. A Perspective From Industry - A Conversation With Richard S. Geary, Ph.D.
(Oligonucleotide Therapeutics Society)
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