Anti-sense Oligonucleotide correction of myotonic dystrophy in HSALR mice.

As I bring forth not got round to writing the be unexhausted installement of Life in a Lab directly to graduating :D, my girlfriend visiting, work at ~s searching and moving back home :'( I reasoning I would post one of the summaries I wrote for the time of my final year which is relative to the a paper that explored the potenital practice of antisense oligonucleotides (ASOs) as a method of treating for myotonic dystrophy. ASOs consist of linked nucleotides (the components that require up our DNA and RNA) that has been designed to purposfully sanction to a targetted stretch of DNA or RNA (RNA is amenable for converting our genes found in DNA into cellular proteins which can then carry extinguished functions). The purpose of this is to one and the other block the production of the protein or to vitiate the RNA completely. Anyway, I sense of possible fulfilment you enjoy it and if you be obliged any questions please feel free to remark.

Two sub-types of myotonic dystrophy remain, DM1 and DM2, both are caused ~ dint of. the expansion of repeat regions in various genes. A CTG triplet-repeat increase in the 3’ untranslated region of DMPK results in DM1, in which place as a CCTG tetra-repeat opening in the first intron of ZNF9 is responsible for DM2 (1). While DM1 and DM2 acquire varying pathologic severity, the mechanism causing them is in the same manner. One of the major pathogenic factors in the one and the other DMs is that the extended RNAs con~ation secondary structures in the nucleus resulting in the aggregation, and deregulation, of RNA binding factors that are involved in other splicing, consequently disrupting mRNA splicing (1).

In 2012, Wheeler (2) risk out to show that 2-methoxyethyl ASOs could be a potential therapeutic option for the management of DM1 via an RNase H-unable to exist without pathway, as the extended DMPK, RNase H and ASOs are at hand in the nucleus (1, 2, 3) and DNA based ASOs have power to exploit the hydrolytic activity of RNase H in-class to degrade target mRNA (4). A peer model was set up using hACTA1 transgenic mice. These mice expressed a human actin transgene that had a 220 CTG expansion in the 3’ untranslated region, resulting in it comely trapped in the nucleus.

Transgenic mice were injected through an ASO that met two terms: 1) successfully lowered hACTA1 expression in web cultures, and 2) few hostile effects in wild type mice. The four ASOs used were; 190401, which bound in the coding region and 5’ to the renew tract, 190403, which bound to the 5’ UTR, 445236 and 445238, the one and the other bound 3’ of the repeat length (fig.1A). Initially they observed whether a single one of the ASOs lowered the condition of hACTA1 mRNA. 190401, 445236 and 445238 were prosperous in producing a cleavage event that resulted in the wearing away of hACTA1, as a reduction of nuclear CUG repeats was observed at the time that the mice were treated with these ASOs, though 190403 failed to degrade the mark mRNA (fig1B, D, E). It was theorised that degeneration of the expanded mRNA would freedom the bound splicing machinery and soothe the myotonic symptoms by allowing other splicing. In order to demonstrate this, the pervading effect of genes that exhibit MBNL1-unable to exist without alternative splicing (Serca1, Ttn, Zasp and Clcn1) was observed in either saline or one of the four ASOs. 190401, 445236 and 445238 luckily increased alternative splicing of the 4 genes, since splicing of the 4 genes in the appearance of 190403 was the same in the manner that in the control (fig.1F-H).

Figure 1 – ASO usage in CUG(220) expanded hACTA1 transgenic mice. Showing the drift ASOs had on hACTA1 mRNA levels (B, D), CUG repeats in the core (E), alternative splicing (F, G) and myotonic grading (F). (A) Shows the covering location of the ASOs and (C) the meaning ASOs targeting other mRNAs had on hACTA1 mRNA (2).

The expression of expanded CUG repeats too resulted in differential expression of the RNA molecules (1, 2). Micro-arrays were used to analyse the transcriptome in the quadriceps of the couple wild type mice and transgenic mice treated with saline, 190401 or 445236. They showed that afterward 4 weeks the transcriptome of ASO treated cells was closer to turbulent type cells than diseased cells. Both the punishment of alternative splicing, and the normalisation of sickly muscle cell’s transcriptome, shows that debasement of mRNA with expanded CUG repeats, via ASOs in a dose-dependent lordship, can lower the myotonic grading of transgenic mouse cells (fig1.H).

Having long-word ASO activity would be more to be desired than short-term activity as otherwise patients would require ASO administration not rarely. Wheeler (2) then investigated the continuance of ASO knockdown by observing what effect ASO 140401 and 445236 had in the rear of stopping ASO treatment for a year. They discerned that the knockdown activity of ASO 140401 had diminished. Whilst hCATA1 mRNA knockdown levels, other splicing normalisation and cleavage of the expanded mRNA were di~ery seen (fig.2A-C), they had increased compared to initially president and cabinet resulting in a return of the myotonic dystrophy symptoms (fig.2D). However the knockdown nimbleness of ASO 445236 had remained difficult to digest throughout the year, with only a corpuscular increase in hCATA1 mRNA levels and splicing defects (fig.2A-D). They too observed that long-term ASO exercise reduced the number of central kernel in myotonic muscle fibres and prevented the decrease in the muscle fibre diameter (fig2E).

Figure 2­ – Levels of hCATA1 mRNA (A), RNase cleavage product (B), alternative splicing defects (C) and myotonic grading (D) of two ASOs after one year of discontinued treatment. (E) How ASO 445236 affected the distribution of nucleus in myotonic muscles after a year (2).Figure 2­ – Levels of hCATA1 mRNA (A), RNase cleavage consequence (B), alternative splicing defects (C) and myotonic grading (D) of couple ASOs after one year of discontinued handling. (E) How ASO 445236 affected the dispensing of nucleus in myotonic muscles in the rear of a year (2).

Wheeler (2) sooner or later confirmed that the mouse model subsistence used was not susceptible to ASOs. They did this by comparing the muscle membrane, ASO hoarding and RNAse H1 mRNA level of the transgenic mice to that of unrefined type mice. They also compared the exercise of ASOs targeting transcripts present in as well-as; not only-but also; not only-but; not alone-but muscle and liver tissue (as the liver cheerfully takes up modified ASOs (5)) and axiom, in both wild type mice and transgenic mice, that muscle copy levels remained the same but were knocked downward in the liver. They also demonstrated that mRNA transcripts held in the centre of the cell are sensitive to RNase cleavage, in the manner that ASOs targeting a nuclear held written copy – Malat – produced a sinewy knockdown.

Finally they demonstrate that ASOs be able to be used to target and injure human DMPK mRNA. In transgenic mice expressing hDMPK CUG(800) ASOs successfully resulted in a decrease of the extended mRNA ready in the muscle, and combined by their other findings suggests that ASOs could have existence used to successfully reduce expanded DMPK transcripts in lusty dystrophy 1. However, whether this translates well from a peer model into human cells requires more distant investigation, as the longevity and force of the ASOs may vary in human cells. Additionally, systemic direction may be much more difficult in humans than in mice and on that account require a novel delivery system.


Meola, G. and Cardani, R., (2014) ‘Myotonic dystrophies: An update without ceasing clinical aspects, genetic, pathology and corpuscular pathomechanisms’ Basis of Disease doi:10.1016/j.bbadis.2014.05.019.

Wheeler, T. M., Leger, A. J., Pandey, S. K., et al (2012) ‘Targeting nuclear RNA with a view to in vivo correction of myotonic dystrophy’ Nature 488 (7409), pp. 111 – 115.

Lorenz, P., Misteli, T., Baker, B. F., et al (2000) ‘Nucleocytoplasmic shutteling: a romance in vivo property of antisense phosphorothioate oligodeoxynucleotides’ Nucleic Acids Res. 28 (2), pp. 582 – 592.

Wu, H., Lima, W. F., Zhang, H., et al (2004) ‘Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs’ J Biol Chem. 279 (17), pp. 17181 – 17189.

Ponnappa, B. C., Israel, Y. (2002) ‘Targetting Kupffer cells through antisense oligonucleotides’ Bioscie. 7:e223-33.

To form the discomfort more clinical, they told the elementary entrepreneurs into longer, weaker claws because leading through the pandemic.

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