Review of DMD multiple exon skipping with Morpholino, Vivo-Morpholino & PPMO by Toshifumi Yokota's group.
Skipping Multiple Exons to Treat DMD-Promises and Challenges.
Aslesh T, Maruyama R, Yokota T.
Biomedicines. 2018 Jan 2;6(1). pii: E1. doi: 10.3390/biomedicines6010001. Review.
Here is an example of an unusual outcome from modifying splicing. Vivo-Morpholinos targeting exon 9 caused skipping of exon 9 and inclusion of exon 10. In normal alternative splicing of this RNA, either exon 9 is included and exon 10 is excised (Pkm1) or exon 9 is excised and exon 10 is excluded (Pkm2).
"Two MOs complementary to the 3′ splice site of exon 9 or one MO complementary to the 5′ splice site of exon 9 were used to block exon 9 inclusion and promote a concurrent inclusion of exon 10 (Fig. 6A and Fig. S3). All three MOs robustly induced skipping of exon 9 and inclusion of exon 10, with MO #2 the most effective (Figs. 6 A–C and Fig. S3). A minor product indicating skipping of both exons 9 and 10 was also observed, as described previously (23, 31)."
Gao Z, Cooper TA. Reexpression of pyruvate kinase M2 in type 1 myofibers correlates with altered glucose metabolism in myotonic dystrophy. PNAS USA. 2013; [Epub ahead of print] doi:10.1073/pnas.1308806110
http://www.pnas.org/content/110/33/13570.full
This image is of a gel retardation assay using RNA with a complementary Morpholino altering its migration.
https://static-content.springer.com/esm/art%3A10.1007%2Fs00705-017-3666-...
This is from the paper:
Phumesin P, Junking M, Panya A, Yongpitakwattana P, Noisakran S, Limjindaporn T, Yenchitsomanus P. Vivo-morpholino oligomers strongly inhibit dengue virus replication and production. Arch Virol. 2017;[Epub ahead of print] doi:10.1007/s00705-017-3666-9
https://link.springer.com/article/10.1007/s00705-017-3666-9
This paper describes setting up systems to determine splicing-related protein-RNA interactions; this might be especially useful for confirming whether a particular putatuve splice-regulatory protein binding site is actually involved in modulating splicing. The oligos reported were not Morpholinos, but I expect this system would be compatible with Morpohlinos.
Hybrid splicing minigene and antisense oligonucleotides as efficient tools to determine functional protein/RNA interactions.
Cywoniuk P, Taylor K, Sznajder ŁJ, Sobczak K.
Sci Rep. 2017 Dec 14;7(1):17587. doi: 10.1038/s41598-017-17816-x.
This is an overview of various antisense drugs, approved, withdrawn, and in clinical trials, along with an introduction to various intisense structural types and modes of action.
Shen X, Corey DR. Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNAs. Nucleic Acid Research. 2017;[Epub ahead of print] doi:/10.1093/nar/gkx1239
https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkx1239/473...
Here's an interesting article on oligo drug impurities, written from a phosphorothioate perspective but with many ideas potentially applicable to Morpholino GMP production.
Capaldi D, Teasdale A, Henry S, Akhtar N, den Besten C, Gao-Sheridan S, Kretschmer M, Sharpe N, Andrews B, Burm B, Foy J. Impurities in Oligonucleotide Drug Substances and Drug Products. Nucleic Acid Therapeutics. December 2017, 27(6): 309-322. https://doi.org/10.1089/nat.2017.0691
http://online.liebertpub.com/doi/full/10.1089/nat.2017.0691
Here is an excerpt from a letter Jim Summerton recently wrote in which he described the reason why Morpholinos are quantitated by measuring UV absorbance at 265 nm in 0.1N HCl.
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Morpholinos are unusual in having exceptionally good base stacking - better than single-stranded DNA and much better than single-stranded RNA. Most likely this exceptional base stacking is a consequence of the lack of ionic charges on the Morpholino backbone and so no electrostatic repulsions act to counter the base stacking - in contrast to the case for nucleic acids.
Because of Morpholinos’ exceptional base stacking they exhibit an exceptional level of hypochromic shift (reduced absorbance values due to base stacking) which varies as a function of the sequence of the nucleobases in any given oligo sequence. At first glance this variability of spectral absorbance as a function of sequence might be expected to preclude precise quantitation of such compounds by their UV absorbance. However, we have overcome this apparent limitation by thoroughly disrupting the base stacking by assessing absorbance in 0.1 N HCl. At this pH cytosines, adenosines, and guanosines are fully ionized - thereby precluding virtually all stacking of the bases. Thus, taking the UV scan in 0.1 N HCl precludes the sequence-dependent hypochromic shifts common to Morpholinos at pH values closer to neutrality where the nucleobases are not ionized.
Further, we picked a specific wavelength, 265 nm, which for each unstacked nuceobase in 0.1 N HCl has a unique absorbance value which is independent of sequence of the Morpholino oligo. By using the 265 nm absorbance values for each of the four different nucleobases we can precisely and reliably calculate the extinction coefficient of any Morpholino oligo independent of its sequence, dependent only on its nucleobase composition.
Therefore, by using 0.1 N HCl as the solvent and reading the absorbance at 265 nm wavelength you can get a precise quantitation of your product independent of nucleobase sequence and without the variable effects of hypochromism arising from sequence-dependent base stacking. That absorbance value precisely reflects the number of each of the four bases in the Morpholino oligo - and does so essentially independent of sequence.
Here is an example where targeting an internal exon of a pre-mRNA caused inclusion of the adjacent intron, an unusual outcome.
Vierstraete J, Willaert A, Vermassen P, Coucke PJ, Vral A, Claes KBM. Accurate quantification of homologous recombination in zebrafish: brca2 deficiency as a paradigm. Sci Rep. 2017;7:16518. doi:10.1038/s41598-017-16725-3
https://www.nature.com/articles/s41598-017-16725-3
The oligo targeted zebrafish brca2 at the e7i7 junction. There are 28 exons in brca2-202, 27 exons in brca2-201 according to Ensembl ENSDARG00000079015 (accessed Nov 28 2017) .
These investigators report enhanced delivery of Vivo-Morpholinos by combining them with Endo-Porter to deliver the oligos into a hard-to-transfect cell line.
Adamo P, Porazinski S, Rajatileka S, Jumbe S, Hagen R, Cheung MK, Wilson I, Ladomery MR. The oncogenic transcription factor ERG represses the transcription of the tumour suppressor gene PTEN in prostate cancer cells. Oncol Lett. 2017 Nov;14(5):5605-5610. doi: 10.3892/ol.2017.6841. Epub 2017 Aug 28.
http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.10...
Anderson JL, Mulligan TS, Shen M-C, Wang H, Scahill CM, Tan FJ, Du SJ, Busch-Nentwich EM, Farber SA. mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay. PLoS Genet. 2017;13(11):e1007105. doi:10.1371/journal.pgen.1007105
