Initial studies using the genetically engineered mice have shown how modified forms of antisense oligonucleotides can target and correct the aberrant RNA splicing by DMI. Mice have been genetically engineered to have skeletal muscle proteins fluoresce different colours dependent on whether they have been successfully treated or not explains Thurman Wheeler, M.D, who adds the fluorescent models allow for monitoring of drug activity with use of a camera to take pictures of living mice.
Myotonic dystrophy is the most common of these disorders which is estimated to affect 1 in 7500 people, it is caused by a CTG repeat expansions in DM protein kinase genes, of which there are no treatments that can alter course of the disease. DM1 has two subtypes, DM1 affects RNA splicing; and DM1 related mutations affect splicing of multiple proteins in skeletal muscle, as well as those involved in insulin metabolism and cardiac function. Expression of mutant DMPK-CUG mRNA in muscle results in delayed relaxation of muscle fiber contraction, progressive muscle wasting, and histopathologic myopathy.
Lack of better experimental tools and appropriate animal models for evaluating new drug candidates represents a significant hurdle in developing therapies for muscular dystrophies. An existing fluorescent protein based system used for cell based studies into a system which can be used in living mice has now been harnessed in a bi-transgenic model mouse of DM1. Skeletal muscle tissue fibers fluoresce green which are affected by aberrant RNA splicing, those with corrected splicing fluoresce red.
Ratios between red and green fluorescence in muscles after candidate treatment provide indicators of how effective the drug is at correcting abnormal splicing. Additionally the team has developed a laser excitation based fluorescence spectroscopy system has also been developed to visualize the fluorescence signals.
The genetically engineered animal models were tested using existing antisense oligonucleotides that target aberrant splicing events. After injection antisense oligonucleotides into muscles the green/red ratio began to increase within 3 days, and persisted over weeks; analysis after 49 days confirmed treatment had corrected aberrant RNA splicing. Additional testing using subcutaneous injections of a different ASO also yielded a therapeutic effect with the green/red ratio increasing within 14 days of the first of four injections and continued to rise following subsequent doses.
Although effective in skeletal muscle ASOs are not ideally suited for treating DM1. Alternative approaches is to use ligand conjugated antisense technology which adds conjugates to ASOs to increase drug uptake, which was tested in the genetically engineered animal to see if it would have any benefits. LICS ASO treatment started to demonstrate therapeutic activity twice as fast, and was effective at half of the dose of unconjugated ASO, which suggests it to be more potent by at least two fold.
The researchers suggests that their data supports further development of LICA technology for treatment of DM1 and other ASO application targeting skeletal muscle. The genetically engineered model may be useful for rapid identification of therapeutic for reducing pathogenicity of CUG transcripts in DM1, new ASO chemistries, conjugates, small molecules, siRNAs, gene therapy vectors for production of antisense RNAs, protein based therapies that rescue aberrant splicing, and gene editing approaches to reduce genomic CTG repeat length or inhibit transcription of CUG repeats.
Long term this technology would be ideal for testing gene editing therapeutic approaches as they become available; quicker identification of therapies and early rejection of failed candidates will help to develop effective treatments for patients being available sooner and at lower costs, explains Dr. Wheeler.
