AMorpholino, also known as aphosphorodiamidate Morpholino oligomer (PMO), is anucleic acid analog whose backbone consists of methylenemorpholine rings linked throughphosphorodiamidate groups, in contrast to the sugars linked throughphosphates found in natural nucleic acids. Syntheticoligomers of Morpholinos are used inmolecular biology to modifygene expression. Morpholinos block access of other molecules to small (~25 base) specific sequences of the base-pairing surfaces ofribonucleic acid (RNA). Morpholinos are used as research tools forreverse genetics byknocking down gene function.
Morpholinos are in development aspharmaceutical therapeutics targeted againstpathogenic organisms such asbacteria[1] orviruses[2] andgenetic diseases.[3] A Morpholino-based drugeteplirsen fromSarepta Therapeutics received accelerated approval from theUS Food and Drug Administration in September 2016 for the treatment of some mutations causingDuchenne muscular dystrophy,[4] although the approval process was mired in controversy. Other Morpholino-based drugsgolodirsen,viltolarsen, andcasimersen (also for Duchenne muscular dystrophy) were approved by the FDA in 2019–2021.[5][6][7]
"Morpholino" is often capitalized to distinguish the nucleic acid type from otherchemicals containing a six-memberedmorpholine ring, but this usage is not consistent across scientific literature. Vivo-Morpholinos and PPMO are modified forms of Morpholinos with chemical groups covalently attached to facilitate entry into cells.
Morpholino oligos were conceived by Summerton (Gene Tools) at AntiVirals Inc. (now Sarepta Therapeutics) and originally developed in collaboration with Weller.[8]
Morpholinos aresyntheticmolecules that are the product of a redesign of naturalnucleic acid structure.[9] Usually 25 bases in length, they bind to complementary sequences of RNA or single-stranded DNA by standard nucleic acidbase-pairing. In terms of structure, the difference between Morpholinos andDNA is that, while Morpholinos have standard nucleic acid bases, those bases are bound to methylenemorpholine rings linked throughphosphorodiamidate groups instead ofphosphates.[9] The figure compares the structures of the two strands depicted there, one of RNA and the other of a Morpholino. Replacement ofanionic phosphates with the uncharged phosphorodiamidate groups eliminates ionization in the usual physiologicalpH range, so Morpholinos in organisms or cells are uncharged molecules. The entire backbone of a Morpholino is made from these modified subunits.
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.[10] Morpholino oligos are often used to investigate the role of a specific mRNA transcript in anembryo. Developmental biologists inject Morpholino oligos into eggs or embryos ofzebrafish,[11] African clawed frog (Xenopus),[12]sea urchin[13] and killifish (F. heteroclitus) producingmorphant embryos, orelectroporate Morpholinos intochick[14] embryos at later development stages. With appropriate cytosolic delivery systems, Morpholinos are effective incell culture.[15][16] Vivo-Morpholinos, in which the oligo is covalently linked to a deliverydendrimer, enter cells when administered systemically in adult animals or in tissue cultures.[17]
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 specificexon to be spliced out of the RNA transcript encoding a protein can help to determine the function of the proteinmoietyencoded by that exon or can sometimes knock down the protein activity altogether. These molecules have been applied to studies in severalmodel organisms, includingmice,zebrafish,frogs andsea urchins.[18] Morpholinos can also modify thesplicing ofpre-mRNA[19] or inhibit the maturation and activity of miRNA.[20] Techniques for targeting Morpholinos to RNAs and delivering Morpholinos into cells have recently been reviewed in a journal article[21] and in book form.[22]
Ineukaryotic organisms, pre-mRNA istranscribed in the nucleus,introns arespliced out, then the mature mRNA is exported from thenucleus to thecytoplasm. The small subunit of theribosome usually starts by binding at the 5' end of the mRNA and is joined there by various othereukaryotic initiation factors, forming the initiation complex. The initiation complex scans along the mRNA strand until it reaches astart codon, and then the large subunit of the ribosome attaches to the small subunit andtranslation of aprotein begins. This entire process is referred to as gene expression; it is the process by which the information in agene, encoded as a sequence of bases inDNA, 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.
Bound to the5'-untranslated region of messenger RNA (mRNA), Morpholinos can interfere with progression of theribosomal initiation complex from the 5' cap to the start codon. This preventstranslation of the coding region of the targetedtranscript (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 byWestern blot.
In 2016 a synthetic peptide-conjugated PMO (PPMO) was found to inhibit the expression ofNew Delhi Metallo-beta-lactamase, an enzyme that many drug-resistant bacteria use to destroy carbapenems.[23][24]
Morpholinos can interfere withpre-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 thenucleophilicadenine 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] andsplice enhancers.[26] Preventing the binding of snRNPU1 (at the donor site) orU2/U5 (at the polypyrimidine moiety and acceptor site) can cause modifiedsplicing, commonly excludingexons 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 ofU11/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 aftergel electrophoresis of RT-PCR products.[19]
Morpholinos have been used to blockmiRNA activity[29][30] and maturation.[20]Fluorescein-tagged Morpholinos combined with fluorescein-specific antibodies can be used as probes forin-situ hybridization to miRNAs.[31] Morpholinos can blockribozyme 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 translationalframeshifts.[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.
Morpholinos have become a standard knockdown tool in animalembryonic systems, which have a broader range of gene expression than adultcells 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 oforganogenesis anddifferentiation are past, with observedphenotypes 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 thewild-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 modified5'-UTR (untranslated region) so that the rescue mRNA contains no target for the Morpholino. The rescue mRNA'scoding 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 includingcell death in thecentral nervous system andsomite tissues of zebrafish embryos.[43] Most of these effects are due to activation ofp53-mediatedapoptosis 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 beenphenocopied 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 observedmorphant 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]
For a Morpholino to be effective, it must be delivered past thecell 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. Amicroinjection 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 iselectroporation, 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 fromendosomes),[16] the Special Delivery system (no longer commercially available, used a Morpholino-DNAheteroduplex and an ethoxylatedpolyethylenimine delivery reagent),[15] electroporation,[47] or scrape loading.[48]
Delivery into adulttissues is usually difficult, though there are a few systems allowing useful uptake of unmodified Morpholino oligos (including uptake intomuscle cells withDuchenne muscular dystrophy[49] or thevascular endothelial cells stressed during balloonangioplasty[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 withcell-penetrating peptides, and, while toxicity has been associated with moderate doses of the peptide conjugates,[51][52] they have been usedin vivo for effective oligo delivery at doses below those causing observed toxicity.[2][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.[17][54] Delivery-enabled Morpholinos, such as peptide conjugates and Vivo-Morpholinos, show promise as therapeutics for viral and genetic diseases.[55]
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