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ON-based drugs approved by the FDA

Here they are so far, the approved oligonucleotide drugs. Note, however, that Morpholinos are oligos but not nucleotides; Exondys 51 (eteplirsen) is the Morpholino drug.

Table 1. ON-based drugs approved by the FDA.

Trade Name; Time to Market; Company
Indication
Vitravene; 1998; Isis (Ionis)
Cytomegalovirus-induced retinitis
Macugen; 2004; Pfizer/Eyetech
Age-related macular degeneration
Kynamro; 2013; Sanofi/Isis (Ionis)
Familial hypercholesterolemia
Exondys 51; 2016; Sarepta Therapeutics
Duchenne muscular dystrophy
Spinraza; 2016; Biogen/Ionis
Spinal muscular atrophy
Defibrotide; 2016; Jazz Pharma
Severe hepatic veno-occlusive disease

From: Sun et al., Molecules 2017;22:1724. doi:10.3390/molecules22101724

Guidelines for morpholino use in zebrafish

If you work with zebrafish or Morpholinos, please read this.

Stainier DYR, Raz E, Lawson ND, Ekker SC, Burdine RD, Eisen JS, Ingham PW, Schulte-Merker S, Yelon D, Weinstein BM, Mullins MC, Wilson SW, Ramakrishnan L, Amacher SL, Neuhauss SCF, Meng A, Mochizuki N, Panula P, Moens CB. Guidelines for morpholino use in zebrafish. PLoS Genet. 2017 Oct 19;13(10):e1007000. doi: 10.1371/journal.pgen.1007000. eCollection 2017 Oct. No abstract available.

Mechanism of uptake of Morpholino oligos in dystrophic (DMD) muscle

This work elucidates the mechanism of uptake of Morpholino oligos in dystrophic (DMD) muscle, including roles for muscle satellite cells and macrophages.

Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle.

Novak JS, Hogarth MW, Boehler JF, Nearing M, Vila MC, Heredia R, Fiorillo AA, Zhang A, Hathout Y, Hoffman EP, Jaiswal JK, Nagaraju K, Cirak S, Partridge TA.

Nat Commun. 2017 Oct 16;8(1):941. doi: 10.1038/s41467-017-00924-7.

https://www.nature.com/articles/s41467-017-00924-7

Mice, C57Bl/10ScSn-mdx/J or C57Bl/10ScSnJ (Bl10 or wild-type) injection

Discussion on other sites:
https://childrensnational.org/news-and-events/childrens-newsroom/2017/ch...
https://www.drugtargetreview.com/news/26773/macrophages-play-critical-ro...
https://musculardystrophynews.com/2017/10/17/study-shows-that-immune-cel...

Confirming Specificity

There are several commonly-used strategies for confirming specificity of a Morpholino oligo. These are the five-mispair experiment, the second non-overlapping 5'-UTR oligo experiment (and the similar two oligos against one mRNA experiments), the mRNA rescue experiment, and my current favorite, the Morpholino-in-a-null-mutant experiment.

I do not recommend the five-mispair control though its use has been commonly reported. Using a five-mispair oligo allows you to do an experiment to determine the effective-and-specific concentration range for your targeted oligo. The targeted oligo and the five-mispair oligo are used in side-by-side experiments. Each oligo is injected over a concentration range, with a group of embryos injected at each concentration. Usually there is a concentration at which the embryos injected with the targeted oligo display a morphant phenotype and the embryos injected with the five-mispair oligo appear wild-type; at a higher concentration, the embryos injected with the five-mispair oligo also display the morphant phenotype. We define the range of concentration between the onset of morphant phenotype with the targeted oligo and the onset of morphant phenotype with the five-mispair oligo as the effective and specific concentration (dose) range. In later experiments, targeted oligo injected within that range should produce specific effects, at least specific enough that if the targeted oligo shares some complementarity with a targetable region of an off-target mRNA but less than the targeted oligo's similarity with the five-mispair oligo then the targeted oligo should not knock down that off-target mRNA. However, it is not always the case that there will be an effective-and-specific concentration window for a given targeted-and-mispair set of oligos. Because of this, many investigators have shifted to using two non-overlapping oligos to demonstrate specificity.

The two non-overlapping 5'-UTR oligos experiment involves comparing the phenotype induced by injection of two different oligos targeted to block translation of the same mRNA. If both sequences induce the same phenotype, that supports the hypothesis that the observed phenotype is due to knockdown of the targeted gene. This has become a very commonly used test of specificity in the zebrafish community.

A variant on this experiment is to use a splice blocking Morpholino to produce the same phenotype as the translation blocking oligo; while this is a nice experiment when it works, it is not always easy to determine which exon to target in order to knock down the activity of the protein and phenocopy the translation blocker's effect. Yet another variation is the two-splice-blocker experiment, in which the same exon is targeted in separate experiments by a splice donor blocker and a splice acceptor blocker. If both of the oligos when used individually cause a clean exon excision, the phenotypes induced by the two oligos should be identical and, again, the hypothesis that the phenotype was triggered by specific excision of the exon is supported. However, activation of a cryptic splice site can confound the experiments using splice-blockers so while identical results indicate specific knockdown, dissimilar results do not preclude that interaction with the pre-mRNA was specific.

Another useful approach using two non-overlapping oligos is to coinject the oligos at reduced doses, so that each oligo is injected at well less than half of the dose which is just sufficient to elicit an altered phenotype when the oligo is used alone. If the coinjection at reduced doses elicits the same phenotype as the higher-dose single-oligo injections, this shows dose synergy and is support for the hypothesis that the phenotype is caused by knockdown of the targeted mRNA and not an unexpected RNA. This is proposed in: Bill BR, Petzold AM, Clark KJ, Schimmenti LA, Ekker SC. A primer for morpholino use in zebrafish. Zebrafish. 2009 Mar;6(1):69-77.
http://www.liebertonline.com/doi/pdfplus/10.1089/zeb.2008.0555
Note that coinjection of oligos targeting the star and guide strands of an miRNA may result in Morpholino dimer formation, as these oligos often have considerable complementarity with each other.

The mRNA rescue is an excellent proof-of-specificity experiment but will not work for some genes. For this experiment, an mRNA is injected which codes for the same protein that the Morpholino oligo knocks down, but the rescue mRNA has a modified Morpholino binding site so that the Morpholino target is not present on the rescue mRNA. For many genes, coinjection of the proper concentration of Morpholino and rescue mRNA results in producing a wild-type embryo, while injecting the Morpholino alone results in a morphant phenotype. However, the timing of the onset of translation is critical for some developmental genes and the early onset of translation resulting from co-injection of Morpholino and rescue mRNA in the early zygote may mess up the developmental process so that the embryos never recapitulate the wild-type phenotype. The location of translation can also be critical, but mRNA injected into an oocyte ends up distributed throughout the embryo.

That said, it is a very satisfying control when it is successful. If you are planning on using mRNA rescues, I recommend that you have your Morpholino targeted in the 5'-UTR without extending into the coding sequence. Some folks have tried rescuing Morpholinos targeted to the coding sequence by taking advantage of the degenerate genetic code to design mismatches into their mRNAs which do not change the amino acids encoded by the rescue mRNA. While this strategy makes sense, we know of several labs that have struggled with the technique without success. We think the chance of success is better if you start with a 5'-UTR Morpholino so that you can retain the original sequence of the coding region and use an irrelevant 5'-UTR sequence for your rescue mRNA.

If you have or can easily make a null mutant, that is now my favorite specificity control. When you put a Morpholino into a genetic null for the oligo's target, you should not see any effect of the oligo (that is, if microinjected into an early zygote, the oligo solution should have the same effect on development as injecting the vehicle solution without the oligo). If you do see additional effects, there are two possibilities: either the oligo is interacting with off-target RNA and causing the effects, or the mutant is not a true null. The Morpholino should also be injected into wild-type zygotes. It is unsurprising if the phenotype of the mutant and the phenotype of the Morpholino-injected organism differ; genetic compensation (change in expression of functionally-related genes) can hide the effect of losing function of a gene, as shown by Didier Stainier's group; a Morpholino can show the effect of the loss-of-function without the confounding effect of genetic compensation (Rossi et al. 2015). By avoiding compensation, the test of the Morpholino in a wild-type background can reveal gene function that would be obscured if only studying the mutant.

Rossi A, Kontarakis Z, Gerri C, Nolte H, Hölper S, Krüger M, Stainier DYR. Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature. 2015 Aug 13;524(7564):230-3. doi: 10.1038/nature14580. Epub 2015 Jul 13.

Which one to use? That depends partially on what you have at hand. I would choose the easiest path first based on your resources. For instance, if you have made a CRISPR mutant, start with injecting the oligo into the mutant. If you can easily make a rescue RNA, try that (but don't be surprised if ectopic expression makes the rescue fail -- try some RNA in an embryo not treated with Morpholino to test the response to ectopic overexpression). If the easiest and least expensive path is to order a non-overlapping Morpholino, that is a reasonable path too. Think about your specific biological system and target gene - can you see a clear advantage to one of these approaches for your system? Might a report of the outcome of a particular specificity control add more value to your publication?

BLAST homology and specificity controls

I have had many discussions about specificity with a Morpholino user who does very careful BLAST analysis of each oligo prior to ordering. I had previously suggested that about 14 bases of complementary (without significant flanking complementarity) is probably an acceptable level of stability for off-target interactions found by BLAST. However, in light of a new report I agreed that a 13-base complementarity at a splice junction is enough to raise concern. Here is an excerpt of my response to her.

-----------------------------------------------

My suggestion that you ignore matches of less than 14 contiguous bases needs revision based on a new paper (1). They claim effects down to 11 bp match. The methods and analysis in the paper look good to me, and so for now I must accept their conclusion that, for their particular targeting sequences, the 11 bp match caused off-target effects.

However, we need to make a distinction between BLAST screening and actual specificity controls. The BLAST improves the chances that the oligo will pass muster when doing specificity controls, but it is the controls that (we hope and strive to) reveal the actual biological effect of an oligo. The BLAST is a good guide, but too much emphasis there can prevent getting on with the real experiment. That said, I think you are doing this well and likely saving money on synthesis of some oligos that would present problems later. A prudent level of pre-synthesis screening probably lies somewhere between accepting 14 and 11 base pairs.

Here is my brief blog post on the paper that has made me suggest being more stringent at the BLAST stage, with a relevant quote from the end of the paper:

Paper on Morpholino specificity and CRISPR-MO combination

Regarding controls, I now favor testing a Morpholino in a null mutant for the oligo's target RNA. In the genetically-compensated background of a null mutant, a Morpholino specific for its RNA target should have no effect and that is good evidence for its specificity. Subsequent testing of the Morpholino in a wild-type (uncompensated) organism reveals the outcome of acute change in gene expression without being obscured by compensation (2). A potential difficulty is that the mutant must be a true null (3). The two-nonoverlapping olgo experiment is my second choice and provides enough information in most circumstances, but it is still possible for several oligos to have the same phenotypic outcome though some or all of that outcome is due to interaction with a off-target RNA (4). RNA rescues are strong, giving good evidence for specificity when they work, but they cannot always work due to confounding effects of ectopic expression (in terms of time or place) of the rescue RNA. I stand by my position that the five-mispair experiment has little value.

Additional note 8 Feb 2018: This zebrafish community paper (5) is relevant to anyone working with Morpholinos and running specificity controls.

(1) Joris M, Schloesser M, Baurain D, Hanikenne M, Muller M, Motte P. Number of inadvertent RNA targets for morpholino knockdown in Danio rerio is largely underestimated: evidence from the study of Ser/Arg-rich splicing factors. Nucleic Acids Res. 2017;[Epub ahead of print] doi:10.1093/nar/gkx638.
(2) Rossi A, Kontarakis Z, Gerri C, Nolte H, Hölper S, Krüger M, Stainier DYR. Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature. 2015 Aug 13;524(7564):230-3. doi: 10.1038/nature14580. Epub 2015 Jul 13.
(3)Novodvorsky P, Watson O, Gray C, Wilkinson RN, Reeve S, Smythe C, Beniston R, Plant K, Maguire R, M K Rothman A, Elworthy S, van Eeden FJ, Chico TJ. klf2ash317 Mutant Zebrafish Do Not Recapitulate Morpholino-Induced Vascular and Haematopoietic Phenotypes. PLoS One. 2015 Oct 27;10(10):e0141611. doi: 10.1371/journal.pone.0141611. eCollection 2015.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141611
"In summary, our work shows that even in the face of clear evidence of a potentially disruptive mutation induced in a gene of interest, it is currently very difficult to be certain that this leads to loss-of-function, and hence to be confident about the role of the gene in embryonic development."
(4) Coffman JA, Dickey-Sims C, Haug JS, McCarthy JJ, Robertson AJ. Evaluation of developmental phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene. BMC Biol. 2004 May 7;2:6.
(5) Stainier DYR, Raz E, Lawson ND, Ekker SC, Burdine RD, Eisen JS, Ingham PW, Schulte-Merker S, Yelon D, Weinstein BM, Mullins MC, Wilson SW, Ramakrishnan L, Amacher SL, Neuhauss SCF, Meng A, Mochizuki N, Panula P, Moens CB. Guidelines for morpholino use in zebrafish. PLoS Genet. 2017 Oct 19;13(10):e1007000. doi: 10.1371/journal.pgen.1007000. eCollection 2017 Oct.
http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.10...

Earlier blog post on using a Morpholino in a mutant (from a conversation with Martin Blum): Validating Morpholino phenotypes with CRISPRs

Paper on Morpholino specificity and CRISPR-MO combination

Number of inadvertent RNA targets for morpholino knockdown in Danio rerio is largely underestimated: evidence from the study of Ser/Arg-rich splicing factors.

Joris M, Schloesser M, Baurain D, Hanikenne M, Muller M, Motte P.

Nucleic Acids Res. 2017;[Epub ahead of print] doi:10.1093/nar/gkx638.

https://academic.oup.com/nar/article/doi/10.1093/nar/gkx638/3984534/Numb...

The end of the Discussion section:
"Therefore, we also think that the only valid control of MO specificity might be the confirmation that its effects are lost in a null background (29). On the other hand, studies using mutants may suffer from masking of the correct phenotype by compensatory mechanisms, as also illustrated here. In such a case, MOs may be useful as they seem unable to trigger the compensatory response, if their specificity is sufficiently proven, and if the 11-nt MIL value is considered. With all these precautions, mutants combined with antisense MOs represent a valid toolbox to elucidate the true function of a gene."

This paper described a set of off-target interactions leading to changes in splicing of unintended transcripts due to partial complementarity of a Morpholino with unexpected splice junctions. In describing these interactions and the changes they trigger, the paper has provided new guidance on the level of off-target homology that should cause concern when vetting new Morpholino designs.

Analysis of potential off-target interactions by BLAST can be guided by the RNA affinity of Morpholinos with complementary subsequences that were found to alter off-target RNA splicing. In particular the predicted Tm of the contiguous complementary motifs that caused off-target splice modulation may be calculated (done below for the motifs in Joris et al. 2017) and compared with Tm calculations for potential off-target interacting sequence motifs found during analysis of new oligo designs. Predicted Tm were calculated using the formula developed by DNASoftware.com, based on Tm studies of Morpholino-RNA hybrids; their Morpholino-RNA Tm calculator is a feature of some of the oligo analysis software available from DNASoftware.com.

Analysis of predicted Tm of splice Morpholino complementary motifs from Figure 4.

ifrd1 splice junction has 15 bases of complementarity with the sMAsrsf5a Morpholino
Contiguous complementary sequence motif in Morpholino: 5'-TTCAGTCTCACCTCT
Predicted Tm: 80.1°C for 10 µM oligo binding to complementary RNA.

g6pca.1 splice junction has 12 bases of complementarity with the sMAsrsf5a Morpholino
Contiguous complementary sequence motif in Morpholino: 5'-TCAGTCTCACCT
Predicted Tm: 71.6°C for 10 µM oligo binding to complementary RNA.

tfpia splice junction has 11 bases of complementarity with the sMAsrsf5a Morpholino
Contiguous complementary sequence motif in Morpholino: 5'-TCAGTCTCACC
Predicted Tm: 64.7°C for 10 µM oligo binding to complementary RNA.

Formatting sequence information for emails (splice modifying oligo designs)

To email sequence information for design of a splice-modifying Morpholino, write introns in lower case and exons in upper case and write the sense sequence 5'-3'. If you show a start codon, put parentheses around it. Here is an example:

GGTGATGTAGTACGC(ATG)GTAGACCATTTTGACAGgtgtacgttattgccgctcgcgcgcgtagcagtgacagtgacgagacgtcagGTTTTGTGCATGCGGCGTAGTAAACAAGgtgccctgcatacgcgctcgtcgaatagctctagatcccaatagcgcaagGGTGGCCCTCGAT

All I need for the design are 10 exonic and 25 intronic bases, like this:

ATTTTGACAGgtgtacgttattgccgctcgcgcgc e1i1
agcagtgacagtgacgagacgtcagGTTTTGTGCA i1e2
AGTAAACAAGgtgccctgcatacgcgctcgtcgaa e2i2
tagctctagatcccaatagcgcaagGGTGGCCCTC i2e3

These can go into a .txt or .doc file. For a pair of pesudoalleles (i.e. for Xenopus laevis oligo designs), give each example of a junction like this:

ATTTTGACAGgtgtacgttattgccgctcgcgcgc e1i1 .S
ATTTTGTCAGgtgtacgttaaagccgctcgcgcgc e1i1 .L

With the sequence information in text the design should be quick.

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