Morpholino

Last updated August 29, 2024

A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. Its molecular structure contains DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Morpholinos block access of other molecules to small (~25 base) specific sequences of the base-pairing surfaces of ribonucleic acid (RNA). Morpholinos are used as research tools for reverse genetics by knocking down gene function.

This article discusses only the Morpholino antisense oligomers, which are nucleic acid analogs. The word "Morpholino" can occur in other chemical names, referring to chemicals containing a six-membered morpholine ring. To help avoid confusion with other morpholine-containing molecules, when describing oligos "Morpholino" is often capitalized as a trade name, but this usage is not consistent across scientific literature. Morpholino oligos are sometimes referred to as PMO (for phosphorodiamidate morpholino oligomer), especially in medical literature. Vivo-Morpholinos and PPMO are modified forms of Morpholinos with chemical groups covalently attached to facilitate entry into cells.

Gene knockdown is achieved by reducing the expression of a particular gene in a cell. In the case of protein-coding genes, this usually leads to a reduction in the quantity of the corresponding protein in the cell. Knocking down gene expression is a method for learning about the function of a particular protein; in a similar manner, causing a specific exon to be spliced out of the RNA transcript encoding a protein can help to determine the function of the protein moiety encoded by that exon or can sometimes knock down the protein activity altogether. These molecules have been applied to studies in several model organisms, including mice, zebrafish, frogs and sea urchins.^[1] Morpholinos can also modify the splicing of pre-mRNA ^[2] or inhibit the maturation and activity of miRNA.^[3] Techniques for targeting Morpholinos to RNAs and delivering Morpholinos into cells have recently been reviewed in a journal article^[4] and in book form.^[5]

Morpholinos are in development as pharmaceutical therapeutics targeted against pathogenic organisms such as bacteria ^[6] or viruses ^[7] and genetic diseases.^[8] A Morpholino-based drug eteplirsen from Sarepta Therapeutics received accelerated approval from the US Food and Drug Administration in September 2016 for the treatment of some mutations causing Duchenne muscular dystrophy,^[9] although the approval process was mired in controversy. Other Morpholino-based drugs golodirsen, viltolarsen, and casimersen (also for Duchenne muscular dystrophy) were approved by the FDA in 2019–2021.^[10]^[11]^[12]

History

Morpholino oligos were conceived by Summerton (Gene Tools) at AntiVirals Inc. (now Sarepta Therapeutics) and originally developed in collaboration with Weller.^[13]

Structure

Morpholinos are synthetic molecules that are the product of a redesign of natural nucleic acid structure.^[14] Usually 25 bases in length, they bind to complementary sequences of RNA or single-stranded DNA by standard nucleic acid base-pairing. In terms of structure, the difference between Morpholinos and DNA is that, while Morpholinos have standard nucleic acid bases, those bases are bound to methylenemorpholine rings linked through phosphorodiamidate groups instead of phosphates.^[14] The figure compares the structures of the two strands depicted there, one of RNA and the other of a Morpholino. Replacement of anionic phosphates with the uncharged phosphorodiamidate groups eliminates ionization in the usual physiological pH range, so Morpholinos in organisms or cells are uncharged molecules. The entire backbone of a Morpholino is made from these modified subunits.

Function

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.^[15] Morpholino oligos are often used to investigate the role of a specific mRNA transcript in an embryo. Developmental biologists inject Morpholino oligos into eggs or embryos of zebrafish,^[16] African clawed frog ( Xenopus ),^[17] sea urchin ^[18] and killifish ( F. heteroclitus ) producing morphant embryos, or electroporate Morpholinos into chick ^[19] embryos at later development stages. With appropriate cytosolic delivery systems, Morpholinos are effective in cell culture.^[20]^[21] Vivo-Morpholinos, in which the oligo is covalently linked to a delivery dendrimer, enter cells when administered systemically in adult animals or in tissue cultures.^[22]

Normal gene expression in eukaryotes

In eukaryotic organisms, pre-mRNA is transcribed in the nucleus, introns are spliced out, then the mature mRNA is exported from the nucleus to the cytoplasm. The small subunit of the ribosome usually starts by binding at the 5' end of the mRNA and is joined there by various other eukaryotic initiation factors, forming the initiation complex. The initiation complex scans along the mRNA strand until it reaches a start codon, and then the large subunit of the ribosome attaches to the small subunit and translation of a protein begins. This entire process is referred to as gene expression; it is the process by which the information in a gene, encoded as a sequence of bases in DNA, is converted into the structure of a protein. A Morpholino can modify splicing, block translation, or block other functional sites on RNA depending on the Morpholino's base sequence.

Blocking translation

Bound to the 5'-untranslated region of messenger RNA (mRNA), Morpholinos can interfere with progression of the ribosomal initiation complex from the 5' cap to the start codon. This prevents translation of the coding region of the targeted transcript (called "knocking down" gene expression). This is useful experimentally when an investigator wishes to know the function of a particular protein; Morpholinos provide a convenient means of knocking down expression of the protein and learning how that knockdown changes the cells or organism. Some Morpholinos knock down expression so effectively that, after degradation of preexisting proteins, the targeted proteins become undetectable by Western blot.

In 2016 a synthetic peptide-conjugated PMO (PPMO) was found to inhibit the expression of New Delhi Metallo-beta-lactamase, an enzyme that many drug-resistant bacteria use to destroy carbapenems.^[23]^[24]

Modifying pre-mRNA splicing

Morpholinos can interfere with pre-mRNA processing steps either by preventing splice-directing small nuclear ribonucleoproteins (snRNP) complexes from binding to their targets at the borders of introns on a strand of pre-mRNA, or by blocking the nucleophilic adenine base and preventing it from forming the splice lariat structure, or by interfering with the binding of splice regulatory proteins such as splice silencers^[25] and splice enhancers.^[26] Preventing the binding of snRNP U1 (at the donor site) or U2/U5 (at the polypyrimidine moiety and acceptor site) can cause modified splicing, commonly excluding exons from the mature mRNA. Targeting some splice targets results in intron inclusions, while activation of cryptic splice sites can lead to partial inclusions or exclusions.^[27] Targets of U11/U12 snRNPs can also be blocked.^[28] Splice modification can be conveniently assayed by reverse-transcriptase polymerase chain reaction (RT-PCR) and is seen as a band shift after gel electrophoresis of RT-PCR products.^[2]

Other applications: blocking other mRNA sites and use as probes

Morpholinos have been used to block miRNA activity^[29]^[30] and maturation.^[3] Fluorescein-tagged Morpholinos combined with fluorescein-specific antibodies can be used as probes for in-situ hybridization to miRNAs.^[31] Morpholinos can block ribozyme activity.^[32] U2 and U12 snRNP functions have been inhibited by Morpholinos.^[33] Morpholinos targeted to "slippery" mRNA sequences within protein coding regions can induce translational frameshifts.^[34] Morpholinos can block RNA editing,^[35] poly(A) tailing^[36] and translocation sequences.^[37] Morpholino activities against this variety of targets suggest that Morpholinos can be used as a general-purpose tool for blocking interactions of proteins or nucleic acids with mRNA.

Specificity, stability and non-antisense effects

Morpholinos have become a standard knockdown tool in animal embryonic systems, which have a broader range of gene expression than adult cells and can be strongly affected by an off-target interaction. Following initial injections into frog or fish embryos at the single-cell or few-cell stages, Morpholino effects can be measured up to five days later,^[38] after most of the processes of organogenesis and differentiation are past, with observed phenotypes consistent with target-gene knockdown. Control oligos with irrelevant sequences usually produce no change in embryonic phenotype, evidence of the Morpholino oligo's sequence-specificity and lack of non-antisense effects. The dose required for a knockdown can be reduced by coinjection of several Morpholino oligos targeting the same mRNA, which is an effective strategy for reducing or eliminating dose-dependent off-target RNA interactions.^[39]

mRNA rescue experiments can sometimes restore the wild-type phenotype to the embryos and provide evidence for the specificity of a Morpholino. In an mRNA rescue, a Morpholino is co-injected with an mRNA that codes for the morphlino's protein. However, the rescue mRNA has a modified 5'-UTR (untranslated region) so that the rescue mRNA contains no target for the Morpholino. The rescue mRNA's coding region encodes the protein of interest. Translation of the rescue mRNA replaces production of the protein that was knocked down by the Morpholino. Since the rescue mRNA would not affect phenotypic changes due to the Morpholino's off-target gene expression modulation, this return to wild-type phenotype is further evidence of Morpholino specificity.^[38] In some cases, ectopic expression of the rescue RNA makes recovery of the wild-type phenotype impossible.

In embryos, Morpholinos can be tested in null mutants to check for unexpected RNA interactions, then used in a wild-type embryo to reveal the acute knockdown phenotype. The knockdown phenotype is often more extreme than the mutant phenotype; in the mutant, effects of losing the null gene can be concealed by genetic compensation.^[40]

Because of their completely unnatural backbones, Morpholinos are not recognized by cellular proteins. Nucleases do not degrade Morpholinos,^[41] nor are they degraded in serum or in cells.^[42]

Up to 18% of Morpholinos appear to induce nontarget-related phenotypes including cell death in the central nervous system and somite tissues of zebrafish embryos.^[43] Most of these effects are due to activation of p53-mediated apoptosis and can be suppressed by co-injection of an anti-p53 Morpholino along with the experimental Morpholino. Moreover, the p53-mediated apoptotic effect of a Morpholino knockdown has been phenocopied using another antisense structural type, showing the p53-mediated apoptosis to be a consequence of the loss of the targeted protein and not a consequence of the knockdown oligo type.^[44] It appears that these effects are sequence-specific; as in most cases, if a Morpholino is associated with non-target effects, the 4-base mismatch Morpholino will not trigger these effects.

A cause for concern in the use of Morpholinos is the potential for "off-target" effects. Whether an observed morphant phenotype is due to the intended knockdown or an interaction with an off-target RNA can often be addressed in embryos by running another experiment to confirm that the observed morphant phenotype results from the knockdown of the expected target. This can be done by recapitulating the morphant phenotype with a second, non-overlapping Morpholino targeting the same mRNA,^[38] by confirmation of the observed phenotypes by comparing with a mutant strain (though compensation will obscure a phenotype in some mutants), by testing the Morpholino in a null mutant background to detect additional phenotypic changes or by dominant-negative methods. As mentioned above, rescue of observed phenotypes by coinjecting a rescue mRNA is, when feasible, a reliable test of specificity of a Morpholino.^[38]^[40]

Delivery

For a Morpholino to be effective, it must be delivered past the cell membrane into the cytosol of a cell. Once in the cytosol, Morpholinos freely diffuse between the cytosol and nucleus, as demonstrated by the nuclear splice-modifying activity of Morpholinos observed after microinjection into the cytosol of cells. Different methods are used for delivery into embryos, into cultured cells or into adult animals. A microinjection apparatus is usually used for delivery into an embryo, with injections most commonly performed at the single-cell or few-cell stage;^[45] an alternative method for embryonic delivery is electroporation, which can deliver oligos into tissues of later embryonic stages.^[46] Common techniques for delivery into cultured cells include the Endo-Porter peptide (which causes the Morpholino to be released from endosomes),^[21] the Special Delivery system (no longer commercially available, used a Morpholino-DNA heteroduplex and an ethoxylated polyethylenimine delivery reagent),^[20] electroporation,^[47] or scrape loading.^[48]

Delivery into adult tissues is usually difficult, though there are a few systems allowing useful uptake of unmodified Morpholino oligos (including uptake into muscle cells with Duchenne muscular dystrophy ^[49] or the vascular endothelial cells stressed during balloon angioplasty ^[50]). Though they permeate through intercellular spaces in tissues effectively, unconjugated PMOs have limited distribution into the cytosol and nuclear spaces within healthy tissues following IV administration. Systemic delivery into many cells in adult organisms can be accomplished by using covalent conjugates of Morpholino oligos with cell-penetrating peptides, and, while toxicity has been associated with moderate doses of the peptide conjugates,^[51]^[52] they have been used in vivo for effective oligo delivery at doses below those causing observed toxicity.^[7]^[53] An octa-guanidinium dendrimer attached to the end of a Morpholino can deliver the modified oligo (called a Vivo-Morpholino) from the blood to the cytosol.^[22]^[54] Delivery-enabled Morpholinos, such as peptide conjugates and Vivo-Morpholinos, show promise as therapeutics for viral and genetic diseases.^[55]

Related Research Articles

An exon is any part of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term exon refers to both the DNA sequence within a gene and to the corresponding sequence in RNA transcripts. In RNA splicing, introns are removed and exons are covalently joined to one another as part of generating the mature RNA. Just as the entire set of genes for a species constitutes the genome, the entire set of exons constitutes the exome.

RNA splicing is a process in molecular biology where a newly-made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). It works by removing all the introns and splicing back together exons. For nuclear-encoded genes, splicing occurs in the nucleus either during or immediately after transcription. For those eukaryotic genes that contain introns, splicing is usually needed to create an mRNA molecule that can be translated into protein. For many eukaryotic introns, splicing occurs in a series of reactions which are catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs). There exist self-splicing introns, that is, ribozymes that can catalyze their own excision from their parent RNA molecule. The process of transcription, splicing and translation is called gene expression, the central dogma of molecular biology.

Xenopus is a genus of highly aquatic frogs native to sub-Saharan Africa. Twenty species are currently described within it. The two best-known species of this genus are Xenopus laevis and Xenopus tropicalis, which are commonly studied as model organisms for developmental biology, cell biology, toxicology, neuroscience and for modelling human disease and birth defects.

Peptide nucleic acid (PNA) is an artificially synthesized polymer similar to DNA or RNA.

Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small fragments of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, molecular cloning and as molecular probes. In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression, or are degradation intermediates derived from the breakdown of larger nucleic acid molecules.

Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

Antisense therapy is a form of treatment that uses antisense oligonucleotides (ASOs) to target messenger RNA (mRNA). ASOs are capable of altering mRNA expression through a variety of mechanisms, including ribonuclease H mediated decay of the pre-mRNA, direct steric blockage, and exon content modulation through splicing site binding on pre-mRNA. Several ASOs have been approved in the United States, the European Union, and elsewhere.

<span class="mw-page-title-main">Spatiotemporal gene expression</span> Activation of genes

Spatiotemporal gene expression is the activation of genes within specific tissues of an organism at specific times during development. Gene activation patterns vary widely in complexity. Some are straightforward and static, such as the pattern of tubulin, which is expressed in all cells at all times in life. Some, on the other hand, are extraordinarily intricate and difficult to predict and model, with expression fluctuating wildly from minute to minute or from cell to cell. Spatiotemporal variation plays a key role in generating the diversity of cell types found in developed organisms; since the identity of a cell is specified by the collection of genes actively expressed within that cell, if gene expression was uniform spatially and temporally, there could be at most one kind of cell.

An organism which has been treated with a morpholino antisense oligo to temporarily knock down expression of a targeted gene is called a morphant.

Sarepta Therapeutics, Inc. is a medical research and drug development company with corporate offices and research facilities in Cambridge, Massachusetts, United States. Incorporated in 1980 as AntiVirals, shortly before going public the company changed its name from AntiVirals to AVI BioPharma soon with stock symbol AVII and in July 2012 changed name from AVI BioPharma to Sarepta Therapeutics and SRPT respectively. As of 2023, the company has four approved drugs.

In molecular biology and genetics, the sense of a nucleic acid molecule, particularly of a strand of DNA or RNA, refers to the nature of the roles of the strand and its complement in specifying a sequence of amino acids. Depending on the context, sense may have slightly different meanings. For example, the negative-sense strand of DNA is equivalent to the template strand, whereas the positive-sense strand is the non-template strand whose nucleotide sequence is equivalent to the sequence of the mRNA transcript.

<span class="mw-page-title-main">Gene Tools</span> Biotechnology company

Gene Tools, LLC is a limited liability company located in Philomath, Oregon, United States that manufactures Morpholino antisense oligos and delivery reagents. Gene Tools was founded in 1997 and began regularly shipping custom-sequence Morpholino oligos in 2000. Current products include Morpholino oligos and Vivo-Morpholinos.

Cell-penetrating peptides (CPPs) are short peptides that facilitate cellular intake and uptake of molecules ranging from nanosize particles to small chemical compounds to large fragments of DNA. The "cargo" is associated with the peptides either through chemical linkage via covalent bonds or through non-covalent interactions.

Xenbase is a Model Organism Database (MOD), providing informatics resources, as well as genomic and biological data on Xenopus frogs. Xenbase has been available since 1999, and covers both X. laevis and X. tropicalis Xenopus varieties. As of 2013 all of its services are running on virtual machines in a private cloud environment, making it one of the first MODs to do so. Other than hosting genomics data and tools, Xenbase supports the Xenopus research community though profiles for researchers and laboratories, and job and events postings.

In molecular biology, exon skipping is a form of RNA splicing used to cause cells to “skip” over faulty or misaligned sections (exons) of genetic code, leading to a truncated but still functional protein despite the genetic mutation.

<span class="mw-page-title-main">Eteplirsen</span> Medication

Eteplirsen is a medication to treat, but not cure, some types of Duchenne muscular dystrophy (DMD), caused by a specific mutation. Eteplirsen only targets specific mutations and can be used to treat about 14% of DMD cases. Eteplirsen is a form of antisense therapy.

<span class="mw-page-title-main">Reverse genetics</span> Method in molecular genetics

Reverse genetics is a method in molecular genetics that is used to help understand the function(s) of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proceeds in the opposite direction to forward genetic screens of classical genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes are controlled by particular genetic sequences.

Solute carrier family 39 member 12 is a protein that in humans is encoded by the SLC39A12 gene.

Toshifumi (Toshi) Yokota is a biomedical scientist and professor of medical genetics at the University of Alberta, also holding the titles of the Friends of Garrett Cumming Research & Muscular Dystrophy Canada Endowed Research Chair and the Henri M. Toupin Chair in Neurological Science. Known for pioneering research in antisense therapy for muscular dystrophy that led to the development of an FDA-approved drug viltolarsen, research interests encompass precision medicine for muscular dystrophy and genetic diseases. Publications exceed 100 refereed papers and patents, with contributions as co-editor to three books in the Methods in Molecular Biology series from Humana Press, Springer-Nature, Roles include fellow of the Canadian Academy of Health Sciences, a member of the editorial boards for numerous journals, a member of the Medical and Scientific Advisory Committee of Muscular Dystrophy Canada, chief scientific officer of OligomicsTx, and a co-founder of the Canadian Neuromuscular Network (CAN-NMD).

Stephen Donald Wilton, also known as Steve Wilton, is an Australian molecular biologist and academic, serving as the Foundation Professor of Molecular Therapy at Murdoch University and adjunct professor at the University of Western Australia (UWA). He also fulfills dual roles as a Director at the Perron Institute for Neurological and Translational Science and deputy director at Murdoch's Centre for Molecular Medicine and Innovative Therapeutics (CMMIT).

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