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Novel Mouse Model Allows Rapid Evaluation of Candidate Drugs for Muscular Dystrophy

Key findings

  • By modifying an existing bichromatic reporter, neurologists at Massachusetts General Hospital created a therapy reporter for myotonic dystrophy type 1 (DM1) for in vivo use
  • The researchers generated a bitransgenic mouse model of DM1 that expresses the modified reporter and confirmed that it provides a highly sensitive and quantitative measure of splicing outcomes based on the ratio of two fluorescent proteins
  • A custom-built fluorescence spectroscopy system rapidly measured splicing outcomes in the mice, with results strongly correlated to those of fluorescence microscopy, but with substantially greater throughput
  • A novel ligand conjugated antisense oligonucleotide, designed to enhance drug uptake into muscle tissue in the bitransgenic mice, was present at concentrations about twofold higher than the parent antisense oligonucleotide after four weeks of treatment
  • It may be possible to modify the therapy reporter for use as a drug development tool for other RNA-processing disorders, such as Duchenne muscular dystrophy and amyotrophic lateral sclerosis

No current treatment alters the disease course of muscular dystrophy. A major obstacle to drug development has been the lack of appropriate animal models for efficiently screening candidate therapeutics. Current methods of measuring pharmacodynamic properties involve biochemical analysis of muscle tissue, which is expensive and time-consuming.

Neurologists at Massachusetts General Hospital have generated a new mouse model that allows convenient and fast estimation of a drug's activity in living animals. The work is described by Assistant Neurologist Thurman M. Wheeler, MD, and colleagues in Nature Communications.

Bichromatic Therapy Reporter

The researchers built on another group's creation of a bichromatic alternative splicing therapy reporter that can express both green fluorescent protein (GFP) and red fluorescent protein (DsRed) from a single alternative splicing event. Aberrant RNA splicing is a hallmark of the cellular dysfunction in myotonic dystrophy type 1 (DM1), the most common muscular dystrophy in adults.

The research team adapted the reporter such that mice would express the human ATP2A1 exon 22 minigene, one of the many transcripts that is misspliced in DM1 muscle. They also restricted expression of the fluorescent proteins to skeletal muscle. The hope was that DsRed would signal in muscle fibers when splicing was appropriate, GFP would signal when splicing was aberrant and the ratio between them would indicate that a therapy was correcting aberrant splicing.

Bitransgenic Mouse Model of DM1

Next, the therapy reporter transgenic mice were bred with human skeletal actin–long repeat (HSALR) transgenic mice, the model currently used to test drugs for DM1. As an initial test of the new mouse model, the researchers injected the gastrocnemius muscles with an antisense oligonucleotide (ASO) known to correct RNA splicing defects in HSALR mice.

Compared with saline-treated muscles, ASO-treated muscles showed an increase in the ratio of DsRed:GFP fluorescence—indicating a treatment effect—by the third day. The increase persisted for several weeks.

Novel In Vivo Fluorescence Spectroscopy System

The researchers constructed a laser excitation–based fluorescence spectroscopy system and compared the DsRed:GFP ratios they obtained to those obtained with fluorescence microscopy. The results were strongly correlated.

However, spectroscopy has a major advantage: speed. With spectroscopy, DsRed:GFP ratios are calculated automatically at the end of each scan, whereas microscopy images must be quantified by hand. Drawing individual regions of interest, subtracting background from each channel, correcting for image exposure time and finally, calculating DsRed:GFP ratios takes several minutes per mouse. Therefore, throughput is substantially lower than with spectroscopy.

Novel Ligand-conjugated Antisense Oligonucleotide

Ligand-conjugated antisense (LICA) chemistry adds specific conjugates to ASOs that are designed to increase drug uptake, which could enable a lower dose and therefore improve tolerability. Dr. Wheeler's group developed a novel LICA-modified ASO and tested it in the bitransgenic mice. The DsRed:GFP ratio in gastrocnemius and lumbar paraspinal muscles increased as early as day seven, after only two doses.

The researchers then compared the LICA oligonucleotide with the unconjugated parent ASO. In mice treated with the LICA oligonucleotide, the DsRed:GFP ratio increased in gastrocnemius muscles by day 14 and in lumbar paraspinal muscles by the seventh day. With the parent ASO, response occurred at Days 28 and 14, respectively. Concentrations of the LICA oligonucleotide in muscle tissue proved to be about twofold higher than those of the parent ASO throughout four weeks of treatment.

Research Implications

The bitransgenic mouse model should be useful for rapid identification of new drugs for DMI, the researchers conclude. It is also well suited for an equally important goal: to reject ineffective drugs at an early stage, before they proceed to costly clinical trials.

The authors add that, considering the sensitivity of spectroscopy, it may be possible to use therapy reporter constructs as biomarkers for other applications. For example, by modifying the minigene, their therapy construct might be useful as a drug development tool for other disorders that are candidates for RNA modulation therapies, such as Duchenne muscular dystrophy and amyotrophic lateral sclerosis.

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