Maksudov F, Kliuchnikov E, Pierson D, Ujwal ML, Marx KA, Chanda A, Barsegov V. Therapeutic Phosphorodiamidate Morpholino Oligonucleotides, an RNA mimic: Physical Properties, Solution Structures, and Folding Thermodynamics. Mol Ther Nucleic Acids. 2023;[Epub] doi:10.1016/j.omtn.2023.02.007
https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S21...(23)00027-6
From: ****
Sent: Wednesday, January 18, 2023 10:17 AM
To: Jon Moulton
Subject: Translation-blocking Vivo-MO reduces gene expression on the level of mRNA?
Hello Jon,
I find that the translation-blocking Vivo-MO is able to significantly reduce the copies on the level of mRNA. I use qPCR and RNAseq to confirm that the mRNA of my target gene is down-regulated by more than 4-fold at 24 hours after injection, and by more than 10-fold at 7 days after injection. Such knockdown effect is impressive, but theoretically, translation blocking MO only blocks translation, which should reduces the level of protein, rather than the level of mRNA. I am just curious about why translation-blocking MO also have effect on the mRNA level.
I also try splice-modifying Vivo-MO, which also knockdown gene expression significantly. But in my case, translation-blocking MO always induces stronger phenotype than the splice-modifying one. I hope this information will be useful to other researchers.
Have a nice day.
Best,
****
-------------------------------------------------
Hi ****,
Binding a Morpholino to an RNA can change the stability of the RNA -- we hypothesize that this is due to change of the secondary structure making the RNA more susceptible to nucleolytic attack. However, we cannot predict what kind of change will occur: when Morpholinos bind RNA there have been reports of degradation, stabilization and lack of effect on RNA lifetime. Because of this we suggest that all translation blocking Morpholinos should be assessed at the level of protein using an immunochemical assay; this gives a more reliable signal.
Thanks for reporting your observation of a decrease in the RNA on binding a Morpholino translation-blocking oligo. May I have your permission to post these two messages on my blog? I would be happy to remove identifying information from your email if you like (let me know your preference). I think this would be useful exchange for others to read.
Thanks!
- Jon
Lim, K.R.Q., Yokota, T. (2018). Invention and Early History of Exon Skipping and Splice Modulation. In: Yokota, T., Maruyama, R. (eds) Exon Skipping and Inclusion Therapies. Methods in Molecular Biology, vol 1828. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8651-4_1
https://link.springer.com/protocol/10.1007/978-1-4939-8651-4_1
Master’s thesis based on microarray analysis of WT, Morphant and rescue fish
Tuttle, Matthew Alan (2017)
In silico analysis of zebrafish leptin-a knockdown gene expression data reveals enrichment for metabolic and developmental pathways including morpholino artifacts
https://etd.ohiolink.edu/apexprod/rws_olink/r/1501/10?clear=10&p10_acces...
Liang H, Li Y, Li M, Zhou W, Chen J, Zhang Z, Yang Y, Ran C, Zhou Z. The effect and underlying mechanism of yeast β-glucan on antiviral resistance of zebrafish against spring viremia of carp virus infection. Front Immunol. 2022 Nov 3;13:1031962. doi: 10.3389/fimmu.2022.1031962. eCollection 2022.
https://www.frontiersin.org/articles/10.3389/fimmu.2022.1031962/full
"Zebrafish larvae (4dpf) were added with 25 nmol/L of CRFB1 and CRFB2 vivo MO (CRFB1 and CRFB2 vivo MO were added together during the experiment), or standard control vivo MO, and then treated with 0.05% β-glucan for 24 h. At 7 dpf, 0.1 MOI SVCV was added. At 9 dpf, zebrafish larvae were collected and SVCV replication in the larvae was measured by qPCR."
The authors of this new paper use an engineered viral vector (AAV) to express a secreted antibody. They make a version where the antibody is out-of-frame and a Morpholino is used to skip an exon, bring the antibody in-frame and switch on its functional expression; the target is a payload of the AAV and the switching molecule is the Morpholino. This use of Morpholinos as switching molecules in engineered systems has great therapeutic potential. In the same paper, they controlled the expression of their AAV cargo with a Vivo-Morpholino after delivery into a mouse. This is a potential of Morpholino oligos that has not yet been talked about much, but I've been excited about for many years; I recall a similar switching technique used with an engineered plasmid long ago, and this traces its roots to the pluc705 splice-actuated reporter plasmid and similar constructs from Ryszard Kole's group in the '90s.
Cripe TP, Hutzen B, Currier MA, Chen CY, Glaspell AM, Sullivan GC, Hurley JM, Deighen MR, Venkataramany AS, Mo X, Stanek JR, Miller AR, Wijeratne S, Magrini V, Mardis ER, Mendell JR, Chandler DS, Wang PY. Leveraging gene therapy to achieve long-term continuous or controllable expression of biotherapeutics. Sci Adv. 2022 Jul 15;8(28):eabm1890. doi: 10.1126/sciadv.abm1890. Epub 2022 Jul 13.
T cells redirected to cancer cells either via a chimeric antigen receptor (CAR-T) or a bispecific molecule have been breakthrough technologies; however, CAR-T cells require individualized manufacturing and bispecifics generally require continuous infusions. We created an off-the-shelf, single-dose solution for achieving prolonged systemic serum levels of protein immunotherapeutics via adeno-associated virus (AAV) gene transfer. We demonstrate proof of principle in a CD19+ lymphoma xenograft model using a single intravenous dose of AAV expressing a secreted version of blinatumomab, which could serve as a universal alternative for CD19 CAR-T cell therapy. In addition, we created an inducible version using an exon skipping strategy and achieved repeated, on-demand expression up to at least 36 weeks after AAV injection. Our system could be considered for short-term and/or repeated expression of other transgenes of interest for noncancer applications.
Single Stranded Fully Modified-Phosphorothioate Oligonucleotides can Induce Structured Nuclear Inclusions, Alter Nuclear Protein Localization and Disturb the Transcriptome In Vitro.
Flynn LL, Li R, Pitout IL, Aung-Htut MT, Larcher LM, Cooper JAL, Greer KL, Hubbard A, Griffiths L, Bond CS, Wilton SD, Fox AH, Fletcher S.
Front Genet. 2022;13:791416. doi:10.3389/fgene.2022.791416
https://www.frontiersin.org/articles/10.3389/fgene.2022.791416/full
Adverse Drug Reactions and Toxicity of the FDA-approved Antisense Oligonucleotide Drugs.
Alhamadani F, Zhang K, Parikh R, Wu H, Rasmussen TP, Bahal R, Zhong XB, Manautou JE. Drug Metab Dispos. 2022 Feb 27:DMD-MR-2021-000418. doi:10.1124/dmd.121.000418. Online ahead of print.
https://dmd.aspetjournals.org/content/early/2022/02/27/dmd.121.000418.long
Absorption, distribution, metabolism, and excretion of FDA-approved antisense oligonucleotide drugs.
Migliorati JM, Liu S, Liu A, Gogate A, Nair S, Bahal R, Rasmussen TP, Manautou JE PhD, Zhong XB. Drug Metab Dispos. 2022 Feb 27:DMD-MR-2021-000417. doi: 10.1124/dmd.121.000417.
https://dmd.aspetjournals.org/content/early/2022/02/27/dmd.121.000417.long
Adverse Drug Reactions and Toxicity of the FDA-approved Antisense Oligonucleotide Drugs.
Alhamadani F, Zhang K, Parikh R, Wu H, Rasmussen TP, Bahal R, Zhong XB, Manautou JE PhD. Drug Metab Dispos. 2022 Feb 27:DMD-MR-2021-000418. doi: 10.1124/dmd.121.000418.
https://dmd.aspetjournals.org/content/early/2022/02/27/dmd.121.000418.long
Goossens R, Aartsma-Rus A. In Vitro Delivery of PMOs in Myoblasts by Electroporation. Methods Mol Biol. 2022;2434:191-205. doi: 10.1007/978-1-0716-2010-6_12.
Antisense oligonucleotides (AONs) are small synthetic molecules of therapeutic interest for a variety of human disease. Their ability to bind mRNA and affect its splicing gives AONs potential use for exon skipping therapies aimed at restoring the dystrophin transcript reading frame for Duchenne muscular dystrophy (DMD) patients. The neutrally charged phosphorodiamidate morpholino oligomers (PMOs) are a stable and relatively nontoxic AON modification. To assess exon skipping efficiency in vitro, it is important to deliver them to target cells. Here, we describe a method for the delivery of PMOs to myoblasts by electroporation. The described protocol for the Amaxa 4D X unit nucleofector system allows efficient processing of 16 samples in one nucleocuvette strip, aiding in high-throughput PMO efficacy screens.
https://link.springer.com/protocol/10.1007/978-1-0716-2010-6_12
