Blogs

Review
Advances in the Study of Heart Development and Disease Using Zebrafish

Daniel R. Brown, Leigh Ann Samsa, Li Qian, Jiandong Liu.
J. Cardiovasc. Dev. Dis. 2016, 3(2), 13; doi: 10.3390/jcdd3020013

http://www.mdpi.com/2308-3425/3/2/13/htm

Keyword search "Morpholino" or go to section "3.1.2. Morpholinos" for a brief discussion of Morpholino efficacy, specificity and controls.

There are a few points in this discussion (3.1.2. Morpholinos) where I disagree, others where I would like to expand on their suggestions.

First, the authors write that Morpholinos are designed to bind to translation-initiation sites of mRNA. We have made many successful oligos targeting upstream of the translation initiation AUG; Morpholinos targeting the 5'-UTR block the scanning complex from completing its journey from the 5'-cap to the start codon.

The authors claim that Morpholinos are slowly degraded by cellular processes. However, when this was explicitly tested no degradation was found [1,2]. Morpholino activity does decrease with time; we hypothesize that when a Morpholino binds an RNA and the RNA is degraded by nucleases, a footprint of RNA is protected bound to the Morpholino. The slow degradation of the footprint and release of single-stranded Morpholino activity explains the persistent low-level splice-modifying activity observed months after Morpholino dosing [3].

In Box 2, the Specificity section mentions several control Morpholinos. In section 4, they list the standard control, the 5-base pair mismatch, and the p53 oligo. I've comments about each.

Standard control: This is an oligo targeting an intronic site in human beta-globin that is mutated in some cases of beta-thalaslemia. It has been extensively used as a negative control for developmental biology, so extensively that some experienced Morpholino users have suggested there is no reason to ever inject it again. I agree, but results from your reviewers may vary.

Five base pair mismatch: This is an old-style specificity control oligo, but I think a better control for specificity is to use a second non-overlapping Morpholino to phenocopy the first oligo's results. Sometimes there is phenotypic bleed-through with the five-mispair oligo. On the whole, I'd be happier if everyone used the second targeting oligo approach.

p53 oligo: this is a very important control, used to determine whether the p53-mediated apoptotic cascade is triggered by loss of a protein [4]. The Morpholino results in a zebrafish paper are stronger if, along with reporting the outcome on injecting a targeting oligo, the results of a p53 oligo co-injected with the targeting oligo are also reported.

[1] Hudziak RM, Barofsky E, Barofsky DF, Weller DL, Huang SB, Weller DD. Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nucleic Acid Drug Dev 1996 Winter;6(4):267-72.

[2] Youngblood DS, Hatlevig SA, Hassinger JN, Iversen PL, Moulton HM. Stability of cell-penetrating Peptide-morpholino oligomer conjugates in human serum and in cells. Bioconjug Chem. 2007 Jan-Feb;18(1):50-60.

[3] Wells DJ. Gene doping: the hype and the reality. Br J Pharmacol. 2008 Jun;154(3):623-31. Epub 2008 Apr 21.

[4] Robu ME, Larson JD, Nasevicius A, Beiraghi S, Brenner C, Farber SA, Ekker SC. p53 activation by knockdown technologies. PLoS Genet. 2007 May 25;3(5):e78. Epub 2007 Apr 10.

Makino S, Mishima Y, Inoue K, Inada T. Roles of mRNA fate modulators Dhh1 and Pat1 in TNRC6-dependent gene silencing recapitulated in yeast. J Biol Chem. 2015 Mar 27;290(13):8331-47. doi: 10.1074/jbc.M114.615088. Epub 2015 Feb 5.
http://www.jbc.org/content/290/13/8331.long

"To address this question, we injected GFP reporter mRNA into zebrafish embryos and then blocked its translation using a morpholino oligomer (ATG MO) that specifically masks the start codon of the GFP ORF."

Webb AB, Lengyel IM, Jörg DJ, Valentin G, Jülicher F, Morelli LG, Oates AC. Persistence, period and precision of autonomous cellular oscillators from the zebrafish segmentation clock. Elife. 2016 Feb 13;5. pii: e08438. doi: 10.7554/eLife.08438.
http://elifesciences.org/content/5/e08438v2

"Monoclonal antibodies were generated to the Ntla protein, the zebrafish T/Brachyury homolog, and to Tbx16, the product of the spadetail locus. 8 μg of Ntla (amino acids 1–261) or Tbx16 peptide (amino acids 232–405) fused to GST was injected into Balb/c mice; sera were screened via ELISA. Each antiserum with a positive signal was further tested for tissue-specific binding in 15-somite stage wild-type and mutant or morpholino-injected embryos."

Someone asked me why some Morpholinos usually cause exon skips and others cause intron inclusions...

U1 and U2 (or U11 and U12) snRNPs mark the positions on pre-mRNA of the splice junctions for the spliceosome. There is a U1 snRNP that binds in the intron near the e2i2 junction and a U2 snRNP that binds on the other side of the intron at the i2e3 junction.

Figure 1

Morpholinos targeting splicing are usually targeted to block the binding sites of these snRNPs. Consider the splice junctions of the second intron. If you block the U2 snRNP binding site with an i1e2 oligo, the spliceosome will match the U1 snRNP at i1e2 with the next available U2 snRNP downstream, which is bound at the i3e3 junction. This splices the e1i1 junction to the i2e3 junction, eliminating i1, e2 and i2.

Figure 2

Now consider what happens if you block the e1i1 junction with a Morpholino. The U1 can't bind to its e1i1 site, but the U2 at the i1e2 site has no other upstream U1 it can be redirected toward to make a splice. In this case, the usual result is for no splicing to occur at i1; that is, you see an i1 inclusion in the mature mRNA.

Figure 3

Because the very first (e1i1) and very last splice sites have no splice sites past them to which splicing can be redirected, blocking them causes a failure to splice (intron inclusion). Blocking any of the internal splice boundaries (i1e2, e2i2, i2e3, etc.) usually causes the adjacent exon to be eliminated from the mature mRNA (or, in the common jargon, causes the exon to be skipped). Here is an illustration of intron inclusion resulting from blocking the last splice junction.

Figure 4

Other things can happen. Activation of cryptic splice sites can cause partial exon excision or partial intron inclusion. Sometimes an oligo causes a double exon skip. Another possible outcome is a failure to splice of an internal intron, causing its inclusion in the mature mRNA. However, the most common outcomes are a intron inclusion for the first and last splice sites and an exon excision (skip) for all other splice junctions.

Figure 5

An entirely different approach to modulating splicing is to block the binding sites of splice regulatory proteins (splice enhancers, splice suppressors). I'm mentioning it here, but not discussing it more.

Typically splice-modifying oligo efficacy is tested with reverse-transcript PCR followed by gel electrophoresis of the PCR product. It can take several primer pairs to determine what happened. Sometimes putting a primer in each of the exons adjacent to the target can reveal a clean excision of the targeted exon. To detect the exon 2 skip in the second figure above, you would typically put a primer in exon 1 and another in exon 3. Other common approaches are to move a primer an additional exon away to look for a double-exon-skip or to place a primer in intronic sequence to detect an intron inclusion. For cryptic splice site activation where the cryptic site is close to the wild-spliced site, sequencing the PCR product can reveal small changes. When choosing primers, be sure that you plan to make RT-PCR products of a hundred bases or so when the exon is missing; that way the RT-PCR product is long enough it can take up plenty of an intercalating fluorescent dye and be readily visible on the gel.

If you do not see an obvious single-exon-skip by looking at the RT-PCR product on an electrophoretic gel, one of these alternative splicing outcomes might be the reason (but the most likely outcome is the single-exon skip). Load the gel lightly and watch for dimming of the wild-spliced band in the MO-treated vs. control lane (normalized against a housekeeping gene band intensity). If the band is over-saturated with too much RNA , it can be harder to see the dimming of the band if some of the transcript is diverted to a different splicing fate, but if you have a lightly-loaded gel then it should be obvious if you lose some band intensity (normalized against a housekeeping gene). If nonsense-mediated decay is occurring, you might not see much or any mass-shifted band from splice-modification, but you should see the wild-spliced band become more dim.

One more time but drawn a bit differently - this is why blocking an internal splice site usually causes exon excision, but blocking the first or last splice site usually causes intron inclusion.

Figure 6

Nice review, a few years old, describing Morpholinos and other antisense as therapeutics.

RNA therapeutics: beyond RNA interference and antisense oligonucleotides.
Kole R, Krainer AR, Altman S.
Nat Rev Drug Discov. 2012 Jan 20;11(2):125-40. doi: 10.1038/nrd3625. Review.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743652/

Sen D, Patel G, Patel SS. Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE. Nucl Acids Res. 2016;[Epub ahead of print] doi:10.1093/nar/gkw098

https://nar.oxfordjournals.org/content/early/2016/02/16/nar.gkw098.full

DNA binds in the protein TWINKLE, but Morpholino is very poor at that binding. No surprise, it's likely due to lack of charge, but this is a nice published example of the low-binding characteristic.

Here's an interesting technique: inhibition of nonsense-mediated decay with oligos. I don't think the antisense oligos were Morpholinos.

Antisense oligonucleotide-directed inhibition of nonsense-mediated mRNA decay.
Nomakuchi TT, Rigo F, Aznarez I, Krainer AR.
Nat Biotechnol. 2015 Dec 14. doi: 10.1038/nbt.3427. [Epub ahead of print]

http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3427.html

Here is a review of tools to manipulate gene expression in the brain. Just before the Conclusion is the section "Use of Morpholinos to Regulate Gene Expression in the Brain".

Walters BJ, Azam AB, Gillon CJ, Josselyn SA, Zovkic IB. Advanced In vivo Use of CRISPR/Cas9 and Anti-sense DNA Inhibition for Gene Manipulation in the Brain. Front Genet. 2016. doi:10.3389/fgene.2015.00362

http://journal.frontiersin.org/article/10.3389/fgene.2015.00362/full

Though I did in the early days of commercial Morpholinos, now I generally don't enter review articles into the Morpholino database. This one came along and I want to keep a record of it, particularly for the list of diseases in Table 1. So, it goes here.

Subbotina E, Koganti SR, Hodgson-Zingman D, Zingman LV. Morpholino-driven gene editing: A new horizon for disease treatment and prevention. Clin Pharmacol Ther. 2015 Oct 16. doi: 10.1002/cpt.276. [Epub ahead of print]

http://onlinelibrary.wiley.com/doi/10.1002/cpt.276/full

Liquori A, Vaché C, Baux D, Blanchet C, Hamel C, Malcolm S, Koenig M, Claustres M, Roux AF. Whole USH2A Gene Sequencing Identifies Several New Deep Intronic Mutations. Hum Mutat. 2015 Nov 2. doi: 10.1002/humu.22926. [Epub ahead of print]

http://onlinelibrary.wiley.com/doi/10.1002/humu.22926/abstract

See online supplement page 12
"Supp. Table S7. In silico analysis of Enhancer motives targeted by AMO in PE 50"
http://onlinelibrary.wiley.com/store/10.1002/humu.22926/asset/supinfo/hu...