Extracellular RNA in Urine Is a Source of Biomarkers for Muscular Dystrophy

In patients with myotonic dystrophy 1 (DM1), disease activity is assessed with muscle biopsies to evaluate pre-mRNA splicing outcomes. However, these biopsies are inconvenient, add cost and can be painful for patients.

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Neurologist Thurman M. Wheeler, MD, and colleagues are in the beginning stages of developing non-invasive liquid biopsies for muscular dystrophy. In Nature Communications, they report the first evidence that extracellular RNA (exRNA) in urine is a source of biomarkers for the disease. This finding has implications for personalized patient monitoring as well as for drug development.

Cells release membrane-encased particles (vesicles) to the extracellular environment not just when they are dying off, but also when they are healthy, as a method of communicating with other cells. Extracellular vesicles in blood and urine contain mRNA and non-coding RNA, collectively termed exRNA. There has been promising research into using exRNA as biomarkers in cancer and some other diseases.

In culture, skeletal muscle cells have been observed to release extracellular vesicles, and some non-coding RNA biomarkers and protein signatures have been identified in the blood of patients with muscular dystrophy.

Examining Alternative mRNA Splice Variants in Urine exRNA

Dr. Wheeler's team compared urine exRNA samples from 40 patients with DM1 against samples from 12 patients with Duchenne muscular dystrophy (DMD), three non-ambulatory patients with Becker muscular dystrophy and 29 unaffected control subjects. They detected 10 alternative splice variants that were unique to the DM1 patients, most of which had been identified previously in muscle biopsies of those patients.

Previous research with DM1 mice highlighted five of those alternative splicing variants as sensitive indicators of therapeutic response.

Composite Biomarker and Predictive Modeling

Next, the researchers performed principal component analysis of the 10 variants and generated a composite splicing biomarker score for each subject. To generate a predictive model for DM1, the researchers pooled composite data from the first 23 DMI patients enrolled, along with the first 22 unaffected subjects, creating a training set.

This revealed wide separation between the patients with DM1 and those who had another form of muscular dystrophy or were unaffected. The composite score was far more powerful than any single splice event.

Data on six DM1 patients and five unaffected controls were used as the validation set. The model was 100% accurate in distinguishing DM1 patients from unaffected subjects. It was also 100% accurate with the next 19 study subjects enrolled (13 DM1 patients and six unaffected controls), for a total of 30 individuals evaluated correctly.

Detection of Drug Activity

One key purpose of a biomarker in muscular dystrophy would be to determine whether a therapy is reaching its molecular target. In patients with DMD, detection of the activity of an antisense oligonucleotide (ASO) currently requires multiple muscle biopsies to examine whether the target exon has been removed from the mRNA.

Dr. Wheeler's group examined urine exRNA from six therapy-naïve patients with DMD and found patient-specific deletion transcripts that corresponded to the specific gene deletion in all six. The researchers then examined urine RNA from two patients with DMD who are being treated with eteplirsen, an exon-skipping ASO. They detected mutation-specific DMD mRNAs in urine that confirmed exon-skipping activity, providing the first non-invasive measurement of eteplirsen target engagement.

Toward the Future

The findings in this study presumably apply to a number of other genetic systemic diseases. The results of DMD have two particularly exciting implications:

1. Urine biomarkers might facilitate the development of novel ASO therapies or target additional exons in DMD
2. Unlike traditional biomarkers, urine splice products could be designed specifically for individual patients with DMD to monitor the splice-shifting activity of ASOs

RNA-based assays won't likely eliminate muscle biopsies in DMD any time soon. The Food and Drug Administration allowed quantification of dystrophin protein, which requires muscle biopsy, as a surrogate marker of drug effect in trials of eteplirsen. However, monitoring of skipped/unskipped DMD splice products in urine over the course of drug treatment might complement splicing analysis of muscle biopsies as better splice-shifting drugs are developed.

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Learn more about the Wheeler Muscular Dystrophy Research Lab

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