New chemical therapeutics of genetic disease by manipulating the transcriptome

Speaker: Masatoshi Hagiwara
Department: Developmental biology
Subject: New chemical therapeutics of genetic disease by manipulating the transcriptome 
Location: Erasmus MC Rotterdam
Date: 3 April 2017
Author: Carolien Bastiaanssen

Professor Hagiwara leads a research group at Kyoto University Graduate School of Medicine Japan. His long-lasting dream is to cure genetic diseases with the compounds he and his colleagues develop.  Nowadays, with the availability of CRISPR-Cas9 as a genome editing technology, it has become relatively easy to manipulate DNA. However, before this discovery it was easier to manipulate RNA than DNA using small and cheap molecules. The research of professor Hagiwara is therefore focused on compounds that influence the splicing of RNA and that can be used in splicing therapies for genetic diseases.

In order to study the effect of different compounds on the splicing of pre-mRNA, professor Hagiwara and his colleagues developed a way to visualize alternative splicing. As a model organism the nematode C. elegans was used with egl-15 as the model gene. This gene encodes a fibroblast growth factor receptor and alternative splicing gives rise to two different isomers containing one of the mutually exclusive exons 5B and 5A. The first isomer, EGL-15(5B),  is essential for viability and the second isomer, EGL-15(5A), is involved in the migration of sex myoblasts. Depending on which exon is present, cells express either GFP or RFP (Figure 1). After this first success professor Hagiwara and his colleagues also developed reporters to for alternative splicing dependent on the developmental stage. Furthermore they succeeded in expressing their reporter system in mice and in mammalian cells.

 alternative splicing reporterFigure 1: Alternative splicing reporter in C. elegans. A) The construct for the alternative-splicing reporter. B) Transgenic C. elegans expressing the aforementioned reporter. From left to right: RFP, GFP, the first two merged, and differential interference contrast (DIC) image. C) Close up of the vulva showing that the vulval muscles express E5A-RFP and not E5B-GFP. Adapted from: Kuroyanagi, H. et al. Transgenic alternative-splicing reporters reveal tissue-specific expression profiles and regulation mechanisms in vivo. Nat Meth 3, 909–915 (2006).

Once they were able to visualize alternative splicing, professor Hagiwara and his colleagues tried to find compounds that could correct for aberrant splicing in order to treat patients with for example familial dysautonomia (FD). This is a hereditary disease caused by mutations in the IkB kinase complex-associated protein (IKAP) gene. In these patients exon 20 is skipped, especially in neurons, and this results in a truncated protein product. FD patients could benefit from a treatment that stimulates exon 20 inclusion. Using a reporter similar to the one shown above, professor Hagiwara and colleagues tested all kinds of compounds in their chemical libraries. One of these compounds increased exon 20 inclusion in cells of FD patients. They named the compound rectifier of aberrant splicing or in short RECTAS. This compound was shown to be effective in cells from FD patients, future studies on FD mouse models are now required to get RECTAS towards clinical trials.

RECTASFigure 2: RECTAS is a small molecule that rescues aberrant splicing in FD cells A) Structure of RECTAS B) Cells that lack exon 20 (thus FD phenotype) express RFP and wildtype cells that do have exon 20 express GFP. DMSO is a negative control and kinetin is a positive control. RECTAS rescues aberrant splicing in FD cells and it does so to a larger extent than kinetin.  C) Quantification of GFP/RFP ratios after treatment with RECTAS or kinetin. Lower concentrations of RECTAS were required to obtain the same effect that was achieved with higher concentrations of kinetin. Source: Yoshida, M. et al. Rectifier of aberrant mRNA splicing recovers tRNA modification in familial dysautonomia. Proc. Natl. Acad. Sci.  112, 2764–2769 (2015).

Another example of a disease where patients can benefit from splicing therapy is Duchenne muscular dystrophy (DMD). This fatal disease is caused by a mutation in the dystrophin gene that results in a lack of the dystrophin protein. The mutation introduces a frameshift, thereby creating a premature stop codon. Thus no dystrophin protein is produced. A milder phenotype of DMD is Becker muscular dystrophy (BMD). Patients with this phenotype also have a mutation in the dystrophin gene, however this mutation does not cause a shift of the reading frame.  Instead the mutation promotes skipping of an exon. Although part of the protein is missing, it is still partially functional therefore BMD patients show less severe symptoms than DMD patients. Thus by treating DMD patients with a compound that causes the mutated exon to be skipped, the symptoms can be drastically reduced. Professor Hagiwara and colleagues found such a compound. Its name is TG003 and it stimulates skipping of  the mutated exon while it does not affect the wildtype exon. More importantly, TG003 did not affect the splicing patterns of the other exons in the dystrophin gene.

All in all, Professor Hagiwara showed that splicing therapies with small molecules can be used to treat FD and Duchenne muscular dystrophy patients. These results are promising, not only for these groups of patients but in the future splicing therapies with small molecules can potentially be used for other genetic diseases as well. Professor Hagiwara tried to explain everything clearly, yet due to his heavy accent I had to pay very close attention to follow the talk. However his passion for his work was obvious and the promising results are of interest for multiple groups at the Erasmus MC who might want to use splicing therapy for the patients they try to help.





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