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Dosing mama mouse i.v. for transplacental delivery of Vivo-Morpholinos to pups

Touma M, Kang X, Gao F, Zhao Y, Cass AA, Biniwale R, Xiao X, Eghbali M, Coppola G, Reemtsen B, Wang Y. Wnt11 regulates cardiac chamber development and disease during perinatal maturation. JCI Insight. 2017 Sep 7;2(17). pii: 94904. doi: 10.1172/jci.insight.94904. eCollection 2017 Sep 7.

https://insight.jci.org/articles/view/94904

Review - Antisense-mediated splice intervention to treat human disease: the odyssey continues

Pitout I, Flynn LL, Wilton SD, Fletcher S.

Antisense-mediated splice intervention to treat human disease: the odyssey continues.

F1000Research 2019,8(F1000 Faculty Rev):710 doi:10.12688/f1000research.18466.1

https://f1000research.com/articles/8-710/v1

Innate immune stimulation by Morpholinos in Xenopus?

The discussion of innate immune stimulation by Morpholinos in Xenopus continues.

Paraiso KD, Blitz IL, Zhou JJ, Cho KWY. Morpholinos Do Not Elicit an Innate Immune Response during Early Xenopus Embryogenesis. Dev Cell. 2019;49(4):643-50. doi:10.1016/j.devcel.2019.04.019
https://www.cell.com/developmental-cell/fulltext/S1534-5807(19)30316-8

Gentsch GE, Spruce T, Owens NDL, Monteiro RS, Smith JC. The Innate Immune Response of Frog Embryos to Antisense Morpholino Oligomers Depends on Developmental Stage, GC Content and Dose. Dev Cell. 2019;49(4):506-7. doi:10.1016/j.devcel.2019.05.004
https://www.cell.com/developmental-cell/fulltext/S1534-5807(19)30375-2

These new papers extend the work presented in:
Gentsch GE, Spruce T, Monteiro RS, Owens NDL, Martin SR, Smith JC. Innate Immune Response and Off-Target Mis-splicing Are Common Morpholino-Induced Side Effects in Xenopus. Dev Cell. 2018;[Epub ahead of print] doi:10.1016/j.devcel.2018.01.022.
http://www.cell.com/developmental-cell/fulltext/S1534-5807(18)30057-1

Their mutants don't show a phenotype and their morphants don't: they suggest that more time is needed for compensation

These investigators suggest a reason their mutants don't show a phenotype and their morphants don't. The mutant embryos have had time enough to compensate for the knockdowns, hiding the phenotype, while the acute knockdown of the Morpholino shows a phenotype during time insufficient for compensation.

Yang C-R, Rajkovic G, Daldello EM, Luong XG, Chen J, Conti M. The RNA binding protein DAZL functions as repressor and activator of maternal mRNA translation during oocyte maturation. bioRxiv. 2019;[preprint] doi:10.1101/598805

https://www.biorxiv.org/content/10.1101/598805v1.full

"Another possible explanation of the divergent observations is that the oocyte does not tolerate acute depletion of DAZL, while it has time to adjust to loss of DAZL during the follicle growth phase that starts in the neonate ovary. Genetic compensation has been shown to be at the basis of differences in phenotypes produced by mutations but not knockdowns. Since we have shown that DAZL functions in partnership with CPEB19, it is possible that this latter RBP would compensate for the loss of DAZL. In this respect, it would be important to determine whether CPEB expression is affected in the DAZL KO and how the translational program is executed in the absence of DAZL in the fully grown oocyte."

Antisense oligonucleotides to treat spinal muscular atrophy (Shift Pharmaceuticals)

Antisense oligonucleotides to treat spinal muscular atrophy
Shift Pharmaceuticals (University of Missouri)
Abstract for Midwest Drug Development Conference, Sept. 30-Oct. 1, 2019. Omaha NE.
https://www.mwdrugdevelopment.com/speakers/oligonucleotides-spinal-muscu...

"We have previously characterized a genetic region upstream of SMN2 exon 7 called Element 1 (E1) that functions as a repressor of SMN2 exon 7 inclusion. The presence of this genetic element reduces the production of the full-length SMN product by promoting the exclusion of exon 7 and the expression of the truncated isoform (SMN-delta7). Molecules that block or inhibit the repressive activity of E1 could be envisioned as potential therapies for SMA is they relieve the repression and allow for high levels of full-length SMN expression from the SMN2 gene. To this end, we have developed an antisense oligonucleotide that anneals to the E1 region using the well-characterized Morpholino chemistry for the molecules’ backbone."

Storage of Morpholinos: refrigerate or room temperature?

Morpholino oligos are completely chemically & biologically (enzymatically) stable sitting in water at 4C or at room temperature as long as the pH is not extreme. However, they undergo physical changes that can affect their biological activity. Chilled storage increases the tendency of the oligos to associate with container walls (this is especially a problem if the oligos undergo freeze-thaw cycles). Once a Morpholino has associated tightly with the wall of a container, it can be difficult or impossible to get the oligo back into solution at neutral pH. Very acidic conditions can protonate the oligo and get it back into the bulk solution phase, but can also damage the Morpholino’s backbone, usually by hydrolyzing off the dimethylamine; this is useful for assessing the presence of Morpholinos on the walls of a dry tube, but not for recovering biological activity of a stock solution that has underwent wall-association. Because of this, we do not recommend storing Morpholinos at 4C.

At room temperature, wall association occurs less but solution-phase aggregation occurs more frequently. This aggregation can decrease the single-stranded antisense activity of a Morpholino solution. However, the biological activity of the solution can usually be recovered by autoclaving. Faced with a choice between the irrecoverable loss of activity though container wall association at low temperature versus the recoverable loss of activity through aggregation at room temperature, we recommend room temperature storage.

Proposed mechanism underlying genetic compensation

Genetic paradox explained by nonsense. Wilkinson MF. Nature. 2019;568:179-180. doi: 10.1038/d41586-019-00823-5
https://www.nature.com/articles/d41586-019-00823-5

Genetic compensation triggered by mutant mRNA degradation. El-Brolosy MA et al. Nature. 2019;568:193-197. doi:10.1038/s41586-019-1064-z
https://www.nature.com/articles/s41586-019-1064-z

PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components. Ma Z et al. Nature. 2019;568:259-263. doi:10.1038/s41586-019-1057-y
https://www.nature.com/articles/s41586-019-1057-y

Ma ZP, Chen J.[Nonsense mutations and genetic compensation response]. Yi Chuan. 2019 May 20;41(5):359-364. doi: 10.16288/j.yczz.19-101. Chinese.
https://www.ncbi.nlm.nih.gov/pubmed/31106771
http://www.cnki.net/kcms/doi/10.16288/j.yczz.19-101.html

Antibody validation by Morpholino

Here is an example of using Morpholinos for antibody validation.

Ratzan W, Rayaprolu V, Killian SE, Bradley R, Kohout SC. The voltage sensing phosphatase (VSP) localizes to the apical membrane of kidney tubule epithelial cells. PLoS ONE. 2019;14(4):e0209056. doi:10.1371/journal.pone.0209056

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0209056

Paper: PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components

Another brick pulled from the wall concealing the mechanism of genetic compensation. Nonsense-mediated decay appears again.

Ma Z, Zhu P, Shi H, Guo L, Zhang Q, Chen Y, Chen S, Zhang Z, Peng J, Chen J. PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components. Nature. 2019;[Epub ahead of print] doi:10.1038/s41586-019-1057-y

https://www.nature.com/articles/s41586-019-1057-y

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