Researchers at the Massachusetts General Hospital (MGH) have developed a fluorescent mouse model of the most common form of muscular dystrophy, myotonic dystrophy type 1 (DM1), which could help scientists find new treatments for the genetic disorder. The team’s initial studies using the genetically engineered mice—in which skeletal muscle proteins fluoresce different colors dependent on whether they have been successfully treated or not—have already shown how modified forms of antisense oligonucleotides (ASO) can target and correct the aberrant RNA splicing caused by DM1.

“This novel fluorescent model allows monitoring of therapeutic drug activity simply by using a camera to take pictures of living mice,” said research lead Thurman Wheeler, M.D., at the MGH department of neurology and an assistant professor of neurology at Harvard Medical School. The researchers report on their studies in Nature Communications, in a paper titled, “Non-invasive monitoring of alternative splicing outcome to identify candidate therapies for myotonic dystrophy type 1.”

Muscular dystrophies are a group of genetic disorders that cause progressive muscle weakness and wasting, the authors explained. Myotonic dystrophy (dystrophia myotonica; DM) is the most common of these disorders in adults, and affects about 1 in 7500 people. The disease is caused by a CTG repeat expansion (CTGexp) in the DM protein kinase (DMPK) gene, but there are no treatments that can alter the course of the disease. DM1 has two subtypes, of which DM1 affects RNA splicing. The DM1-related mutation affects the splicing of multiple proteins in skeletal muscle, as well as those that are involved in insulin metabolism and cardiac function. Expression of mutant DMPK-CUGexp mRNA in muscle results in myotonia (delayed relaxation of muscle fiber contraction), histopathologic myopathy, and progressive muscle wasting,” the researchers stated.

New therapies for muscular dystrophies and better experimental tools to help find new treatments are critically needed, but the lack of appropriate animal models for evaluating new drug candidates is a key hurdle. “Current methods of measuring drug activity involve biochemical analysis of muscle tissue, which is expensive, time-consuming, and may require large numbers of animals to achieve the necessary statistical power,” the team commented.

Dr. Wheeler’s team has now harnessed an existing fluorescent protein-based system used for cell-based studies, into a system that can be used in living animals to light up skeletal muscle tissue proteins. In this bi-transgenic mouse model of DM1 skeletal muscle tissue fibers that are affected by aberrant RNA splicing fluoresce green, while those with corrected splicing fluoresce red. “By crossing an established mouse model of myotonic dystrophy type 1 with one that expresses either a red or green fluorescent protein in muscle, depending on the splicing of a target RNA sequence, we developed a model in which muscles appear mostly green before treatment and transition to mostly red after successful treatment,” noted Dr. Wheeler.

The ratio between the red and green fluorescence in the animals’ muscles after candidate treatments have been given indicates how effective the drug is at correcting the abnormal splicing. The team also developed a laser-excitation-based fluorescence spectroscopy system to visualize the fluorescence signals.

The team first tested their model using an existing antisense oligonucleotide (ASO) that targets the aberrant splicing events. ASOs are modified nucleic acid molecules that bind to RNA. “In DM1 transgenic mice, ASOs that target the RNA-mediated disease process can reverse RNA-mis-splicing, eliminate myotonia and slow myopathic progression …” the authors explained. Following injection of the ASO into the muscle, the red/green ratio began to increase within three days, and persisted over weeks. Muscle tissues analyzed after 49 days confirmed that the treatment had corrected the aberrant RNA splicing. A second series of tests using subcutaneous injections of a different ASO known to correct RNA splicing in the original DM1 mouse model also resulted in a therapeutic effect, with the red/green ratio increasing within 14 days of the first of four injections, and continuing to rise following subsequent doses.

ASOs aren’t ideally suited for treating DM1, the authors noted. Although they are effective in skeletal muscle, “drug activity is less robust than in other tissues such as the liver, which may be due to a combination of poor tissue bioavailability and insufficient potency.” An alternative approach is to use ligand-conjugated antisense (LICA) technology, which adds conjugates to the ASOs to increase drug uptake. The team tested a LICA-modified ASO in the mouse model, to see whether it would work in skeletal muscle and have any benefits overtreatment using unconjugated ASO.

The LICA ASO started to demonstrate therapeutic activity twice as fast as the unconjugated ASO, and was effective at half the dose, “suggesting it is at least two-fold more potent,” the researchers stated.  “Our data support further development of LICA technology for the treatment of DM1 and other for ASO applications targeting skeletal muscle,” they concluded. “This model will be useful for rapid identification of candidate therapeutics for reducing pathogenicity of CUGexp transcripts in DM1, including new ASO chemistries and conjugates, small molecules, short interfering RNAs (siRNAs), gene therapy vectors for the production of antisense RNAs, protein-based therapies that rescue aberrant splicing, and gene editing approaches that reduce the genomic CTG repeat length or inhibit transcription of CUGexp repeats.”

“Our results support further development of LICA technology for the treatment of DM1. In addition to new ASOs, other treatment strategies such as small-molecule candidate drugs, siRNAs, and protein-based therapies also could be tested using this model,” Dr. Wheeler said. “Long term it would be ideal for testing gene-editing therapeutic approaches, as they become available. Faster identification of promising therapies and early rejection of failed candidates will help make effective treatments available to patients sooner and at lower developmental costs.”


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