Urine contains extracellular RNA (exRNA) markers of urogenital cancers. However, the capacity of genetic material in urine to identify systemic diseases is unknown. Here we describe exRNA splice products in human urine as a source of biomarkers for the two most common forms of muscular dystrophies, myotonic dystrophy (DM) and Duchenne muscular dystrophy (DMD). Using a training set, RT-PCR, droplet digital PCR, and principal component regression, we identify ten transcripts that are spliced differently in urine exRNA from patients with DM type 1 (DM1) as compared to unaffected or disease controls, form a composite biomarker, and develop a predictive model that is 100% accurate in our independent validation set. Urine also contains mutation-specific DMD mRNAs that confirm exon-skipping activity of the antisense oligonucleotide drug eteplirsen. Our results establish that urine mRNA splice variants can be used to monitor systemic diseases with minimal or no clinical effect on the urinary tract.

The present study aims to determine the effect of AMPK on etoposide-induced apoptosis of cancer cells. Our results revealed that etoposide induced AMPK activation in prostate C4-2 cancer cells, an event that was attenuated by ATM siRNA. In A549 cells that lack LKB1, AMPK was unable to be activated by etoposide, which was restored by introduction of LKB1. Likewise, silencing LKB1 in C4-2 cells impaired AMPK activation. Finally, etoposide displayed a potent pro-apoptotic effect in cancer cells with functional LKB1 and AMPK. Thus, our results establish a linear relationship of ATM, LKB1 and AMPK in response to the DNA damage drug.

During drug development, tissue samples serve as indicators of disease activity and pharmacodynamic responses. Reliable non-invasive measures of drug target engagement will facilitate identification of promising new treatments. Here we develop and validate a novel bi-transgenic mouse model of myotonic dystrophy type 1 (DM1) in which expression of either DsRed or GFP is determined by alternative splicing of an upstream minigene that is mis-regulated in DM1. Using a novel in vivo fluorescence spectroscopy system, we show that quantitation of the DsRed/GFP ratio provides an accurate estimation of splicing outcomes in muscle tissue of live mice that nearly doubles throughput over conventional fluorescence imaging techniques. Serial in vivo spectroscopy measurements in mice treated with a C16 fatty acid ligand conjugated antisense (LICA) oligonucleotide reveal a dose-dependent therapeutic response within seven days, confirm a several-week duration of action, and demonstrate a two-fold greater target engagement as compared to the unconjugated parent oligonucleotide.