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Targeting alternative splicing to upregulate protein expression

Potential application in antisense therapeutics: upregulation of targeted protein expression

Here is a new paper demonstrating techniques for targeting steric-blocking antisense oligos to alternatively-spliced transcripts undergoing NMD to alter the splicing and recover transcripts encoding useful proteins. This is a technique for antisense upregulation of a protein activity.

Lim KH, Han Z, Jeon HY, Kach J, Jing E, Weyn-Vanhentenryck S, Downs M, Corrionero A, Oh R, Scharner J, Venkatesh A, Ji S, Liau G, Ticho B, Nash H, Aznarez I. Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression. Nat Commun. 2020;11(1):3501. doi:10.1038/s41467-020-17093-9

Retention of the last intron

Here are a few publications addressing Morpholinos and retention of the last intron.

See the section: Retention of wnt11b Intron 4 Recapitulates the Failure to Form Somites in tra2b Morphants
Dichmann DS, Walentek P, Harland RM. The Alternative Splicing Regulator Tra2b Is Required for Somitogenesis and Regulates Splicing of an Inhibitory Wnt11b Isoform. Cell Rep. 2015 Jan 21. pii: S2211-1247(14)01099-7. doi: 10.1016/j.celrep.2014.12.046. [Epub ahead of print].

Forcing a terminal intron inclusion:
Shchelkunova A, Ermolinsky B, Boyle M, Mendez I, Lehker M, Martirosyan KS, Kazansky AV. Tuning of Alternative Splicing - Switch From Proto-Oncogene to Tumor Suppressor. Int J Biol Sci. 2013;9(1):45-54. doi:10.7150/ijbs.5194.

Blocking regulatory elements to increase intron retention:
Parra M, Yeo G, Conboy JG. Intron Retention Mechanisms That Regulate SF3B1 and Mitoferrin Gene Expression during Late Erythropoiesis. Blood. 2016;128:1200.

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

PMO-based miRNA site blocking oligo (SBO) mediated utrophin upregulation in mdx mice, a therapeutic approach for Duchenne Muscular Dystrophy (DMD)

Safety of eteplirsen in Duchenne Muscular Dystrophy post-Cardiac transplantation

Open-Label Evaluation of Eteplirsen in Male Patients With Duchenne Muscular Dystrophy Amenable to Exon 51 Skipping vs Untreated Control: PROMOVI Trial

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.

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.

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:'-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.


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