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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:

Giuseppe Ronzitti. Let’s switch on AAV! Sci Transl Med. 2020;12(579) doi:10.1126/scitranslmed.aba9016 (editorial)

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

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

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

"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)."

Morpholino assay in plasma: HPLC-MS/MS Coupled with Solid Phase Micro-Extraction

Two Quantitative Assays for the Phosphorodiamidate #Morpholino Oligomer (PMO) SRP-4045 in Mouse Plasma Using HPLC-MS/MS Coupled with Solid Phase Micro-Extraction. Poster at ASMS 2017.

A broader view: Approved Gene Therapy Products 2019

Development and Clinical Translation of Approved Gene Therapy Products for Genetic Disorders.

Shahryari1 A, Jazi MS, Mohammadi S, Nikoo HR, Nazari Z, Hosseini ES, Burtscher I, Mowla SJ, Lickert H.

Front Genet. 25 September 2019. doi:10.3389/fgene.2019.00868

Paper: Site Selective Antibody-Oligonucleotide Conjugation via Microbial Transglutaminase.

This paper describes using a microbial transglutamase to put an azide group in a peptide onto the terminal carboxylic acid of an antibody protein, then using Cu-free click chemistry to affix an antisense oligo at that site. They didn't use Morpholinos, but in principle one could.

Huggins IJ, Medina CA, Springer AD, van den Berg A, Jadhav S, Cui X, Dowdy SF. Site Selective Antibody-Oligonucleotide Conjugation via Microbial Transglutaminase. Molecules. 2019 Sep 10;24(18). pii: E3287. doi: 10.3390/molecules24183287.

Paper on 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.


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