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Paper in delivery in cultures: In Vitro Validation of Phosphorodiamidate Morpholino Oligomers.

Aung-Htut MT, McIntosh CS, West KA, Fletcher S, Wilton SD. In Vitro Validation of Phosphorodiamidate Morpholino Oligomers. Molecules. 2019;24(6):2922 doi:0.3390/molecules24162922

cell culture: human dermal fibroblasts, human myoblasts
electroporation (nucelofection), Lipofectin, Lipofectamine, Endo-Porter

Delay in development and behavioural abnormalities in the absence of p53 in zebrafish

Delay in development and behavioural abnormalities in the absence of p53 in zebrafish.
Elabd S, Jabeen NA, Gerber V, Peravali R, Bourdon JC, Kancherla S, Vallone D, Blattner C.
PLoS One. 2019 Jul 19;14(7):e0220069. doi: 10.1371/journal.pone.0220069. eCollection 2019.

This paper reports changes in developmental timing and behavior of p53-/- mutant embryos. While previous reports had suggested that p53 is dispensable for zebrafish development, the story might not be so simple. Previously a p53 knockdown was expected to produce an essentially wild-type embryo, but on the basis of this new work the results of Morpholino co-injection with p53 Morpholinos should be interpreted cautiously for contributions to the observed phenotype due to lack of p53. Still, given the gross abnormalities of an embryo in which the p53 apoptotic response is triggered by a knockdown, eliminating these abnormalities by a concurrent p53 knockdown is likely to remain a valuable technique for revealing gene function.

Example of specificity control using a Morpholino in its mutant

This paper is a nice example of Morpholino specificity control by using a Morpholino in its corresponding mutant.

Chambers BE, Gerlach GF, Clark EG, Chen KH, Levesque AE, Leshchiner I, Goessling W, Wingert RA. Tfap2a is a novel gatekeeper of nephron differentiation during kidney development. Development. 2019 Jun 3. pii: dev.172387. doi: 10.1242/dev.172387. [Epub ahead of print]

See especially figure S4:

Targeting mitochondrially-encoded circular RNAs in the cytosol: mitochondrial protein uptake suppression

Here is an interesting report of Morpholinos targeting mitochondrially-encoded circular RNAs in the cytosol to prevent RNA-chaperoned protein uptake into the mitochondria.

Liu X, Wang X, Li J, Hu S, Deng Y, Yin H, Bao X, Zhang QC, Wang G, Wang B, Shi Q, Shan G. The identification of mecciRNAs and their roles in mitochondrial entry of proteins. bioRxiv. 2019;[preprint] doi:10.1101/668665

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.

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

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

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

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.

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

"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.

"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."


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