Extracellular microRNAs exhibit sequence-dependent stability and cellular release kinetics

Multiple studies have described extracellular microRNAs (ex-miRNAs) as being remarkably stable despite the hostile extracellular environment, when stored at 4oC or lower. Here we show that many ex-miRNAs are rapidly degraded when incubated at 37oC in the presence of serum (thereby simulating physiologically relevant conditions). Stability varied widely between miRNAs, with half-lives ranging from similar to 1.5 hours to more than 13 hours. Notably, ex-miRNA half-lives calculated in two different biofluids (murine serum and C2C12 mouse myotube conditioned medium) were highly similar, suggesting that intrinsic sequence properties are a determining factor in miRNA stability. By contrast, ex-miRNAs associated with extracellular vesicles (isolated by size exclusion chromatography) were highly stable. The release of ex-miRNAs from C2C12 myotubes was measured over time, and mathematical modelling revealed miRNA-specific release kinetics. While some ex-miRNAs reached the steady state in cell culture medium within 24 hours, the extracellular level of miR-16 did not reach equilibrium, even after 3 days in culture. These findings are indicative of miRNA-specific release and degradation kinetics with implications for the utility of ex-miRNAs as biomarkers, and for the potential of ex-miRNAs to transfer gene regulatory information between cells

Assessment of RT-qPCR Normalization Strategies for Accurate Quantification of Extracellular microRNAs in Murine Serum

<div><p>Extracellular microRNAs (miRNAs) are under investigation as minimally-invasive biomarkers for a wide range of disease conditions. We have recently shown in a mouse model of the progressive muscle-wasting condition Duchenne muscular dystrophy (DMD) that a set of highly elevated serum miRNAs reflects the regenerative status of muscle. These miRNAs are promising biomarkers for monitoring DMD disease progression and the response to experimental therapies. The gold standard miRNA detection methodology is Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR), which typically exhibits high sensitivity and wide dynamic range. Accurate determination of miRNA levels is affected by RT-qPCR normalization method and therefore selection of the optimal strategy is of critical importance. Serum miRNA abundance was measured by RT-qPCR array in 14 week old mice, and by individual RT-qPCR assays in a time course experiment spanning 48 weeks. Here we utilize these two datasets to assess the validity of three miRNA normalization strategies (a) normalization to the average of all Cq values from array experiments, (b) normalization to a stably expressed endogenous reference miRNA, and (c) normalization to an external spike-in synthetic oligonucleotide. Normalization approaches based on endogenous control miRNAs result in an under-estimation of miRNA levels by a factor of ∼2. An increase in total RNA and total miRNA was observed in dystrophic serum which may account for this systematic bias. We conclude that the optimal strategy for this model system is to normalize to a synthetic spike-in control oligonucleotide.</p></div

Serum time course of putative reference miRNA abundance.

<p>Male C57Bl/10, <i>mdx</i> and Pip6e-PMO treated <i>mdx</i> mice were sacrificed at various ages and serum miRNA levels determined by small RNA TaqMan RT-qPCR. (A) miR-1, (B) miR-133a and (C) miR-206 abundance was normalized to an external spike-in control. All miRNA expression data were normalized to the mean of the 8 week old C57Bl/10 group. Values are mean +/− SEM, n = 3–8.</p

Correlation of Cq values between endogenous miRNAs and the external spike-in control for time course samples.

<p>Plot of raw Cq values from the time course study for cel-miR-39 against (A) miR-16, (B) miR-31, and (C) miR-223.</p

Effect of normalization strategy on global serum microRNA abundance.

<p>(A) Key results from NormFinder algorithm. The most stable normalizer (the average of all Cq values) is ranked as 1, the least stable (miR-133b) is ranked as 124. Stability scores and standard error of the mean are shown. (B) Raw Cq data for candidates reference miRNAs and RNA spike-in control. High ranked NormFinder candidates are shown. miR-133b which is known to be highly up-regulated in <i>mdx</i> serum is shown for comparison. (C) C57 vs <i>mdx</i> expression ratios as determined by the average Cq method (x-axis) or normalized to the RNA spike (y-axis).</p

Aggregate microRNA raw Cq data.

<p>Tukey box plots showing raw Cq data aggregated over all time points and separated by experimental condition for each miRNA assayed by small RNA TaqMan RT-qPCR. n numbers for each experimental group are indicated.</p

Non-uniform dystrophin re-expression after CRISPR-mediated exon excision in the dystrophin/utrophin double-knockout mouse model of DMD

Duchenne muscular dystrophy (DMD) is the most prevalent inherited myopathy affecting children, caused by genetic loss of the gene encoding the dystrophin protein. Here we have investigated the use of the Staphylococcus aureus CRISPR-Cas9 system and a double-cut strategy, delivered using a pair of adeno-associated virus serotype 9 (AAV9) vectors, for dystrophin restoration in the severely affected dystrophin/utrophin double-knockout (dKO) mouse. Single guide RNAs were designed to excise Dmd exon 23, with flanking intronic regions repaired by non-homologous end joining. Exon 23 deletion was confirmed at the DNA level by PCR and Sanger sequencing, and at the RNA level by RT-qPCR. Restoration of dystrophin protein expression was demonstrated by western blot and immunofluorescence staining in mice treated via either intraperitoneal or intravenous routes of delivery. Dystrophin restoration was most effective in the diaphragm, where a maximum of 5.7% of wild-type dystrophin expression was observed. CRISPR treatment was insufficient to extend lifespan in the dKO mouse, and dystrophin was expressed in a within-fiber patchy manner in skeletal muscle tissues. Further analysis revealed a plethora of non-productive DNA repair events, including AAV genome integration at the CRISPR cut sites. This study highlights potential challenges for the successful development of CRISPR therapies in the context of DMD

A preclinical model of peripheral T‐cell lymphoma GATA3 reveals DNA damage response pathway vulnerability

Abstract Peripheral T‐cell lymphoma (PTCL) represents a rare group of heterogeneous diseases in urgent need of effective treatments. A scarcity of disease‐relevant preclinical models hinders research advances. Here, we isolated a novel mouse (m)PTCL by serially transplanting a lymphoma from a germinal center B‐cell hyperplasia model (Cγ1‐Cre Blimp1fl/fl) through immune‐competent mice. Lymphoma cells were identified as clonal TCRβ+ T‐helper cells expressing T‐follicular helper markers. We also observed coincident B‐cell activation and development of a de novo B‐cell lymphoma in the model, reminiscent of B‐cell activation/lymphomagenesis found in human PTCL. Molecular profiling linked the mPTCL to the high‐risk “GATA3” subtype of PTCL, showing GATA3 and Th2 gene expression, PI3K/mTOR pathway enrichment, hyperactivated MYC, and genome instability. Exome sequencing identified a human‐relevant oncogenic β‐catenin mutation possibly involved in T‐cell lymphomagenesis. Prolonged treatment responses were achieved in vivo by targeting ATR in the DNA damage response (DDR), a result corroborated in PTCL cell lines. This work provides mechanistic insight into the molecular and immunological drivers of T‐cell lymphomagenesis and proposes DDR inhibition as an effective and readily translatable therapy in PTCL