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Vivo-Morpholino uptake by zebrafish larvae at 4dpf

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."

Vivo-Morpholinos as switching molecules for virally-delivered targets in mice

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.

https://www.science.org/doi/10.1126/sciadv.abm1890

Head-to-head comparisons, Morpholinos and Phosphorothioates

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

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

Pharmacol & Tox of FDA-approved antisense

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

Electroporation protocol: Amaxa 4D X unit nucleofector system

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

Analysis of an apparent mutant/morphant disagreement

Barthelson K, Baer L, Dong Y, Hand M, Pujic Z, Newman M, Goodhill GJ, Richards RI, Pederson SM, Lardelli M. Zebrafish Chromosome 14 Gene Differential Expression in the fmr1 (h u2787) Model of Fragile X Syndrome. Front Genet. 2021 May 31;12:625466. doi: 10.3389/fgene.2021.625466. eCollection 2021.

https://www.frontiersin.org/articles/10.3389/fgene.2021.625466/full

Interesting analysis of an apparent mutant/morphant disagreement finding no genetic compensation experimentally.

"Kok et al. (2015) summarised the puzzling, frequent discordance between phenotypes caused by gene mutations compared to morphant phenotypes caused by reduction of gene expression due to injection of morpholinos. Rossi et al. (2015), then described the phenomenon of “genetic compensation” (now referred to as “transcriptional adaptation” Kontarakis and Stainier, 2020) as contributing to this discordance. As elaborated in a subsequent paper from that laboratory (El-Brolosy et al., 2019), non-sense-medicated decay (NMD) of transcripts with premature termination codons can (in a manner independent of protein feedback loops) increase the abundance of transcripts of genes with homologous sequences that, presumably, are partially functionally redundant and ameliorate the effects of the mutation. The discovery of this phenomenon raises questions regarding the definition of “null” mutant phenotypes and reveals that reducing gene expression using morpholinos may, in some cases, provide more focussed functional effects at the molecular level that are simpler to interpret than those caused by mutations inducing NMD."

"Despite the apparent NMD of fmr1hu2787 transcripts, and the reported milder developmental phenotype of fmr1hu2787 homozygotes relative to individuals in which the function of this gene is suppressed using morpholinos, we did not see evidence for transcriptional adaptation by increased transcription of genes possessing sequences with homology to fmr1, at least at 2 dpf. This illustrates possible variability of the occurrence of the recently discovered transcriptional adaptation mechanism, and that more research is required to understand the factors modulating it."

Generating Zebrafish RNA-Less Mutant Alleles by Deleting Gene Promoters with CRISPR/Cas9

A frameshift mutation did not phenocopy the corresponding Morpholino. To avoid transcriptional adaptation (genetic compensation), the authors made mutants with deleted promoters, eliminating the NMD transcript that would trigger transcriptional adaptation.

Generating Zebrafish RNA-Less Mutant Alleles by Deleting Gene Promoters with CRISPR/Cas9.
Kumari P, Sturgeon M, Bonde G, Cornell RA. Methods Mol Biol. 2022;2403:91-106. doi: 10.1007/978-1-0716-1847-9_8.
https://link.springer.com/protocol/10.1007%2F978-1-0716-1847-9_8

N=1 clinical trials (paper)

Preparing n-of-1 Antisense Oligonucleotide Treatments for Rare Neurological Diseases in Europe: Genetic, Regulatory, and Ethical Perspectives.
Synofzik M, van Roon-Mom WMC, Marckmann G, van Duyvenvoorde HA, Graessner H, Schüle R, Aartsma-Rus A. Nucleic Acid Ther. 2021 Sep 29. doi: 10.1089/nat.2021.0039. Online ahead of print.
https://pubmed.ncbi.nlm.nih.gov/34591693/

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