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Nucleic Acids as Therapeutic Targets and Agents
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
Morpholinos are also being used in the development of diagnostic assays. For example, fluorescein-tagged Morpholinos combined with fluorescein-specific antibodies can be used as probes for in situ hybridization to miRNAs.
Approaches to Studying Polycystic Kidney Disease in Zebrafish
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Reverse genetics, including knockdown and knockout, are widely used in studying the function of genes. In the zebrafish field, as the RNAi technique is not working, we use morpholinos (MO), which are DNA analogues and knockdown gene function by blocking translation or splicing of the target transcripts. Although this technique has been recently replaced by the CRISPR/Cas9-mediated gene knockout technique, due to the off-target issue by the MO, it is still widely used. For example, if we want to block the maternal deposit of a gene transcript and the maternal-zygotic mutant is difficult to obtain, we use MO. To avoid the nonspecific effects of the MO, we should follow the guidelines.20 Basically, the criteria for a reliable MO are that the phenotypes caused by MO knockdown should phenocopy the mutants and can be rescued by its mRNA overexpression.
Drug Discovery: From Hits to Clinical Candidates
Published in Divya Vohora, The Third Histamine Receptor, 2008
Sylvain Celanire, Florence Lebon, Holger Stark
To improve brain receptor occupancy, scientists at J&J PRD have pursued their search of novel H3 antagonists belonging to their established dibasic pharmacophore and investigated linker rigidification (see also section Recent Advance in H3 Receptor Ligand Computer-Aided Drug Design). Indeed, SAR around the conformational restricted iso-propyl piperidin-4-oxyphenyl framework provided highly potent H3 antagonists, as exemplified by compounds 48a (hH3 pKi 8.7; pA2 9.3) and 48b (hH3 pKi 9.2; pA2 9.9) [90, 91]. On the basis of previous investigations, the profile of morpholino derivative 48a (JNJ-7737782) was more detailed, with high selectivity on a panel of 50 targets (CEREP, <18% inhibition at 1 μM) but with a lower binding affinity for the rH3R (pKi 7.8). JNJ-7737782 has demonstrated in vivo efficacy in a rat Electroencephalogram (EEG) EEG model of wakefulness, supporting the therapeutic use of H3 antagonists in sleep/wake disorders and narcolepsy.
Antisense Oligonucleotide Therapy for Ophthalmic Conditions
Published in Seminars in Ophthalmology, 2021
Kevin Ferenchak, Iris Deitch, Rachel Huckfeldt
The phosphodiester backbone of single-stranded RNA and AON can easily degrade in a cell, which limited the practical applications of antisense therapy for many years. Since they were first described, modifications have developed, which make AON viable in vivo with a longer half-life, more resistant to degradation, and more likely to be taken up by cells. These modifications include changes such as the first generation phosphorothioate (PS) backbone, which improved nuclear uptake.23 A phosphoramidate morpholino has been investigated to improve stability, but its viability may be limited by quicker clearance.24,25 Common second generation modifications are a 2ʹO-methoxy ethyl (2MOE) or 2ʹO-methyl (2OME) group attached to the sugar residues of the PS backbone, which further increase stability and cellular uptake.26,27
zHSF1 modulates zper2 expression in zebrafish embryos
Published in Chronobiology International, 2018
Lucas Mennetrier, Tatiana Lopez, Benoist Pruvot, Nadhir Yousfi, Olivier Armant, Hanae Hazhaz, Vincent Lhuissiez, Carmen Garrido, Johanna Chluba
Gene knockdown experiments were performed using morpholino-modified antisense oligonucleotides (Gene Tools, LLC; Philomath OR, USA). All morpholinos were labeled with Lissamine or Fluorescein to facilitate screening of successfully injected eggs. For the hsf1-mo, we used the sequence: 5′-CACGGAGAGTTTAGTGATGATTTCT-3′. The specificity of this morpholino was already demonstrated by other groups (Evans et al. 2007; Etard et al. 2015; Tucker et al. 2011). As control, we used the standard nonsense control morpholino (control-mo) with the sequence 5′-CCTCTTACCTCAGTTACAATTTATA-3′. The morpholinos were injected into one to two cell stage embryos at 250 µM concentration. This was the lowest effective concentration and was determined by titration experiments followed by morphological examination. After injection, only eggs with uniform fluorescence were used for the experiments. The embryos were raised up to 48 hpf at 28 °C.
Construction of nanostructured DNA harbouring phosphorodiamidate morpholino oligonucleotide for controlled tissue distribution in mice
Published in Journal of Drug Targeting, 2018
Yosuke Takahashi, Yuki Araie, Daiki Nomura, Yuki Takahashi, Kohei Sano, Hideo Saji, Yoshinobu Takakura, Makiya Nishikawa
Conjugates, including vivo-morpholino, have been developed to improve the pharmacokinetics of PMO. However, in general, conjugation increases the difficulty of synthesis and variability in structural properties. Alternatively, the method used in the present study only requires ODNs that can be synthesised in high purity at low cost without direct modification of PMO. Moreover, Ferguson et al. [35] reported that vivo-morpholino had cellular toxicity derived from its cationic charges. Our method requires no cationic charges, and PMO/tripodna did not have toxic effects on RAW264.7 cells. Our previous studies using nanostructured DNA and DNA hydrogels showed that these DNA-based systems induced few adverse effects in mice [23,36]. Therefore, our method may be a simpler, less expensive and less toxic method to improve the pharmacokinetics of PMO.