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Presentations/posters from the 2020 MDA conference involving Morpholinos

Long-term Safety & Efficacy of Golodirsen in Male Patients with Duchenne Muscular Dystrophy (DMD) Amenable to Exon 53 Skipping
https://mdaconference.org/node/936

PMO-based miRNA site blocking oligo (SBO) mediated utrophin upregulation in mdx mice, a therapeutic approach for Duchenne Muscular Dystrophy (DMD)
https://mdaconference.org/node/1000

Safety of eteplirsen in Duchenne Muscular Dystrophy post-Cardiac transplantation
https://mdaconference.org/node/913

Open-Label Evaluation of Eteplirsen in Male Patients With Duchenne Muscular Dystrophy Amenable to Exon 51 Skipping vs Untreated Control: PROMOVI Trial
https://mdaconference.org/node/1088

Shift Pharmaceuticals Orphan Drug: Morpholino for Spinal Muscular Atrophy

Shift Pharmaceuticals receives an Orphan Drug designation from the FDA for their Morpholino to treat Spinal Muscular Atrophy.

https://smauk.org.uk/blog/treatments-research/shift-pharmaceuticals-rece...

https://www.accessdata.fda.gov/scripts/opdlisting/oopd/detailedIndex.cfm...

Golodirsen: First Approval. Morpholino drug for DMD (exon 53 skipper)

Golodirsen: First Approval.
Heo YA.
Drugs. 2020 Feb 6. doi: 10.1007/s40265-020-01267-2. [Epub ahead of print] Review.
https://dx.doi.org/10.1007/s40265-020-01267-2

This is an FDA-approved Morpholino drug that causes skipping of exon 53 of human dystrophin, used for the treatment of Duchenne muscular dystrophy.

Available fluorochromes attached to Morpholinos

I like carboxyfluorescein. It is bright, it doesn’t alter how the oligo behaves, and most labs already have a fluorescein cube (filters and dichroic mirror) for their fluorescent microscopes. The down side is that fluorescein photobleaches, so you should minimize its exposure to intense light and only observe it with the microscope when you are ready to take pictures and turn off the lamp soon. Carboxyfluorescein emits green.

Gene Tools Blue is not as bright as fluorescein but it too doesn’t alter how the oligo behaves. The problem is that it is a non-standard fluorochrome and it is expensive to purchase a cube optimized for the fluor (the wavelengths for excitation and emission are here: https://www.gene-tools.com/custom_morpholinos_controls_endmodifications#3'-GeneToolsBlue). A DAPI cube will produce some fluorescent signal, but it is not very good. Gene Tools blue emits blue.

Lissamine, also called Rhodamine B, is a bright fluorochrome and it doesn’t bleach easily but it makes the oligos less water soluble, which can be a practical problem. Lissamine emits red.

https://www.gene-tools.com/custom_morpholinos_controls_endmodifications#...

A reversible RNA on-switch using a Vivo-Morpholino to controls gene expression of AAV-delivered therapeutics in vivo

Zhong G, Wang H, He W, Li Y, Mou H, Tickner ZJ, Tran MH, Ou T, Yin Y, Diao H, Farzan M. A reversible RNA on-switch that controls gene expression of AAV-delivered therapeutics in vivo. Nat Biotechnol. 2019;[Epub ahead print] doi:10.1038/s41587-019-0357-y
Vivo-Morpholino used to control activity of an engineered ribozyme

Press release:
https://www.eurekalert.org/pub_releases/2019-12/sri-nta122419.php

Giuseppe Ronzitti. Let’s switch on AAV! Sci Transl Med. 2020;12(579) doi:10.1126/scitranslmed.aba9016 (editorial)
https://stm.sciencemag.org/content/12/529/eaba9016

FDA approves golodirsen (a Morpholino drug)

Sarepta Therapeutics Announces FDA Approval of VYONDYS 53™ (golodirsen) Injection for the Treatment of Duchenne Muscular Dystrophy (DMD) in Patients Amenable to Skipping Exon 53
https://investorrelations.sarepta.com/news-releases/news-release-details...
https://www.fda.gov/news-events/press-announcements/fda-grants-accelerat...

Paper comparing splice-modifying efficacy of Morpholinos and 2'-O-Me phosphorothioates

McIntosh CS, Aung-Htut MT, Fletcher S, Wilton SD. Removal of the Polyglutamine Repeat of Ataxin-3 by Redirecting pre-mRNA Processing. Int J Mol Sci. 2019;20(21):5434. doi:10.3390/ijms20215434

https://www.mdpi.com/1422-0067/20/21/5434/htm

Excerpt from abstract:
"Here, we describe improved efficiency in the removal of the toxic polyglutamine tract of ataxin-3 in vitro using phosphorodiamidate morpholino oligomers, when compared to antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone. Significant downregulation of both the expanded and non-expanded protein was induced by the morpholino antisense oligomer, with a greater proportion of ataxin-3 protein missing the polyglutamine tract. With growing concerns over toxicity associated with long-term administration of phosphorothioate oligonucleotides, the use of a phosphorodiamidate morpholino oligomer may be preferable for clinical application. These results suggest that morpholino oligomers may provide greater therapeutic benefit for the treatment of spinocerebellar ataxia type 3, without toxic effects."

Excerpt from introduction:
"Here, we describe efficient removal of the CAG containing exon 10 to produce a truncated ataxin-3
protein, lacking the polyglutamine tract, an isoform reported by Toonen et al. (2017) to be functionally
active [8]. Our study shows that by using the PMO chemistry, not only is exon 10 skipping enhanced
at the RNA level, but also significant downregulation of the protein with higher number of glutamine
repeats and an increase in production of the truncated protein is observed, when compared to the use
of the 20-Me PS AO chemistry. With robust splice switching efficiency and an established long-term
safety profile, the PMO oligomers described here are presented as lead pre-clinical candidates to treat
SCA3 patients."

Protein interrelationship shown by synergy of Morpholinos to different mRNAs

Oprişoreanu A-M, Smith HL, Arya S, Webster R, Zhong Z, Wehner D, Cardozo MJ, Becker T, Talbot K, Becker CG. Interaction of Axonal Chondrolectin with Collagen XIXa1 Is Necessary for Precise Neuromuscular Junction Formation. Cell Rep. 2019;29:1082-98. doi:10.1016/j.celrep.2019.09.033

https://www.cell.com/cell-reports/fulltext/S2211-1247(19)31221-5

"Using established (Hilario et al., 2010; Zhong et al., 2012) morpholinos, we found that injection of low concentrations of either morpholino alone elicited no or only weak changes of motor axon growth (Figures 6A and 6B). However, combining these morpholinos at the same concentrations led to synergistic effects on stalling of axons at the horizontal myoseptum. Of note, total morpholino load was kept at the same level for all experiments by adding non-specific control morpholino where appropriate. This indicates a genetic interaction between chodl and col19a1(Figures 6C and 6D)."

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