Implementation of whole-exome sequencing for pharmacogenomics profiling and exploring its potential clinical utilities.

Whole-exome sequencing (WES) is widely used in clinical settings; however, the exploration of its use in pharmacogenomic analysis remains limited. Our study compared the variant callings for 28 core absorption, distribution, metabolism and elimination genes by WES and array-based technology using clinical trials samples. The results revealed that WES had a positive predictive value of 0.71-0.92 and a sensitivity of single-nucleotide variants between 0.68 and 0.95, compared with array-based technology, for the variants in the commonly targeted regions of the WES and PhamacoScan™ assay. Besides the common variants detected by both assays, WES identified 200-300 exclusive variants per sample, totalling 55 annotated exclusive variants, including important modulators of metabolism such as rs2032582 (ABCB1) and rs72547527 (SULT1A1). This study highlights the potential clinical advantages of using WES to identify a wider range of genetic variations and enabling precision medicine.

A Phase Ib Study of the DNA-PK Inhibitor Peposertib Combined with Neoadjuvant Chemoradiation in Patients with Locally Advanced Rectal Cancer.

PURPOSE: Peposertib-an orally administered DNA-dependent protein kinase inhibitor-has shown potent radiosensitization in preclinical models. This dose-escalation study (NCT03770689) aimed to define the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of peposertib plus capecitabine-based chemoradiotherapy (CRT) and assessed its safety and efficacy in locally advanced rectal cancer. PATIENTS AND METHODS: Patients were treated for 5 to 5.5 weeks with 50- to 250-mg peposertib once daily, capecitabine 825 mg/m2 twice daily, and radiotherapy (RT), 5 days per week. Following clinical restaging (8 weeks after CRT completion), patients with clinical complete response (cCR) could opt for surveillance. Total mesorectal excision was recommended upon incomplete response (IR). RESULTS: Nineteen patients were treated with peposertib at doses of 50 mg (n = 1), 100 mg, 150 mg, and 250 mg (n = 6 each). Dose-limiting toxicities occurred in one out of five (100 mg), one out of six (150 mg), and three out of six (250 mg) evaluable patients. Peposertib ≤150 mg once daily was tolerable in combination with CRT. After 8 weeks of treatment with peposertib and CRT, the cCR was 15.8% (n = 3). Among the three patients with cCR, two underwent surgery and had residual tumors. Among the 16 patients with IR, seven underwent surgery and had residual tumors; five of the remaining nine patients opted for consolidative chemotherapy. The combined cCR/pathologic complete response (pCR) rate was 5.3% (n = 1, 100 mg cohort). CONCLUSIONS: Peposertib did not improve complete response rates at tolerable dose levels. The study was closed without declaring the MTD/RP2D.

Synthetic lethality prediction in DNA damage repair, chromatin remodeling and the cell cycle using multi-omics data from cell lines and patients.

Discovering synthetic lethal (SL) gene partners of cancer genes is an important step in developing cancer therapies. However, identification of SL interactions is challenging, due to a large number of possible gene pairs, inherent noise and confounding factors in the observed signal. To discover robust SL interactions, we devised SLIDE-VIP, a novel framework combining eight statistical tests, including a new patient data-based test iSurvLRT. SLIDE-VIP leverages multi-omics data from four different sources: gene inactivation cell line screens, cancer patient data, drug screens and gene pathways. We applied SLIDE-VIP to discover SL interactions between genes involved in DNA damage repair, chromatin remodeling and cell cycle, and their potentially druggable partners. The top 883 ranking SL candidates had strong evidence in cell line and patient data, 250-fold reducing the initial space of 200K pairs. Drug screen and pathway tests provided additional corroboration and insights into these interactions. We rediscovered well-known SL pairs such as RB1 and E2F3 or PRKDC and ATM, and in addition, proposed strong novel SL candidates such as PTEN and PIK3CB. In summary, SLIDE-VIP opens the door to the discovery of SL interactions with clinical potential. All analysis and visualizations are available via the online SLIDE-VIP WebApp.

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.

Exon skipping induces uniform dystrophin rescue with dose-dependent restoration of serum miRNA biomarkers and muscle biophysical properties.

Therapies that restore dystrophin expression are presumed to correct Duchenne muscular dystrophy (DMD), with antisense-mediated exon skipping being the leading approach. Here we aimed to determine whether exon skipping using a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) conjugate results in dose-dependent restoration of uniform dystrophin localization, together with correction of putative DMD serum and muscle biomarkers. Dystrophin-deficient mdx mice were treated with a PPMO (Pip9b2-PMO) designed to induce Dmd exon 23 skipping at single, ascending intravenous doses (3, 6, or 12 mg/kg) and sacrificed 2 weeks later. Dose-dependent exon skipping and dystrophin protein restoration were observed, with dystrophin uniformly distributed at the sarcolemma of corrected myofibers at all doses. Serum microRNA biomarkers (i.e., miR-1a-3p, miR-133a-3p, miR-206-3p, miR-483-3p) and creatinine kinase levels were restored toward wild-type levels after treatment in a dose-dependent manner. All biomarkers were strongly anti-correlated with both exon skipping level and dystrophin expression. Dystrophin rescue was also strongly positively correlated with muscle stiffness (i.e., Young's modulus) as determined by atomic force microscopy (AFM) nanoindentation assay. These data demonstrate that PPMO-mediated exon skipping generates myofibers with uniform dystrophin expression and that both serum microRNA biomarkers and muscle AFM have potential utility as pharmacodynamic biomarkers of dystrophin restoration therapy in DMD.

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

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 TCRß+ 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 ß-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.

Circulating miR-181 is a prognostic biomarker for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a relentless neurodegenerative disease of the human motor neuron system, where variability in progression rate limits clinical trial efficacy. Therefore, better prognostication will facilitate therapeutic progress. In this study, we investigated the potential of plasma cell-free microRNAs (miRNAs) as ALS prognostication biomarkers in 252 patients with detailed clinical phenotyping. First, we identified, in a longitudinal cohort, miRNAs whose plasma levels remain stable over the course of disease. Next, we showed that high levels of miR-181, a miRNA enriched in neurons, predicts a greater than two-fold risk of death in independent discovery and replication cohorts (126 and 122 patients, respectively). miR-181 performance is similar to neurofilament light chain (NfL), and when combined together, miR-181 + NfL establish a novel RNA-protein biomarker pair with superior prognostication capacity. Therefore, plasma miR-181 alone and a novel miRNA-protein biomarker approach, based on miR-181 + NfL, boost precision of patient stratification. miR-181-based ALS biomarkers encourage additional validation and might enhance the power of clinical trials.

Macrophage Activation Status Rather than Repolarization Is Associated with Enhanced Checkpoint Activity in Combination with PI3Kγ Inhibition.

Suppressive myeloid cells mediate resistance to immune checkpoint blockade. PI3Kγ inhibition can target suppressive macrophages, and enhance efficacy of immune checkpoint inhibitors. However, how PI3Kγ inhibitors function in different tumor microenvironments (TME) to activate specific immune cells is underexplored. The effect of the novel PI3Kγ inhibitor AZD3458 was assessed in preclinical models. AZD3458 enhanced antitumor activity of immune checkpoint inhibitors in 4T1, CT26, and MC38 syngeneic models, increasing CD8+ T-cell activation status. Immune and TME biomarker analysis of MC38 tumors revealed that AZD3458 monotherapy or combination treatment did not repolarize the phenotype of tumor-associated macrophage cells but induced gene signatures associated with LPS and type II INF activation. The activation biomarkers were present across tumor macrophages that appear phenotypically heterogenous. AZD3458 alone or in combination with PD-1-blocking antibodies promoted an increase in antigen-presenting (MHCII+) and cytotoxic (iNOS+)-activated macrophages, as well as dendritic cell activation. AZD3458 reduced IL-10 secretion and signaling in primary human macrophages and murine tumor-associated macrophages, but did not strongly regulate IL-12 as observed in other studies. Therefore, rather than polarizing tumor macrophages, PI3Kγ inhibition with AZD3458 promotes a cytotoxic switch of macrophages into antigen-presenting activated macrophages, resulting in CD8 T-cell-mediated antitumor activity with immune checkpoint inhibitors associated with tumor and peripheral immune activation.

Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy.

The absence of the dystrophin protein in Duchenne muscular dystrophy (DMD) results in myofiber fragility and a plethora of downstream secondary pathologies. Although a variety of experimental therapies are in development, achieving effective treatments for DMD remains exceptionally challenging, not least because the pathological consequences of dystrophin loss are incompletely understood. Here we have performed proteome profiling in tibialis anterior muscles from two murine DMD models (mdx and mdx52) at three ages (8, 16, and 80 weeks of age), all n = 3. High-resolution isoelectric focusing liquid chromatography-tandem MS (HiRIEF-LC-MS/MS) was used to quantify the expression of 4974 proteins across all 27 samples. The two dystrophic models were found to be highly similar, whereas multiple proteins were differentially expressed relative to WT (C57BL/6) controls at each age. Furthermore, 1795 proteins were differentially expressed when samples were pooled across ages and dystrophic strains. These included numerous proteins associated with the extracellular matrix and muscle function that have not been reported previously. Pathway analysis revealed multiple perturbed pathways and predicted upstream regulators, which together are indicative of cross-talk between inflammatory, metabolic, and muscle growth pathways (e.g. TNF, INFγ, NF-κB, SIRT1, AMPK, PGC-1α, PPARs, ILK, and AKT/PI3K). Upregulation of CAV3, MVP and PAK1 protein expression was validated in dystrophic muscle by Western blot. Furthermore, MVP was upregulated during, but not required for, the differentiation of C2C12 myoblasts suggesting that this protein may affect muscle regeneration. This study provides novel insights into mutation-independent proteomic signatures characteristic of the dystrophic phenotype and its progression with aging.

Olaparib and durvalumab in patients with germline BRCA-mutated metastatic breast cancer (MEDIOLA): an open-label, multicentre, phase 1/2, basket study.

BACKGROUND: Poly (ADP-ribose) polymerase inhibitors combined with immunotherapy have shown antitumour activity in preclinical studies. We aimed to assess the safety and activity of olaparib in combination with the PD-L1-inhibitor, durvalumab, in patients with germline BRCA1-mutated or BRCA2-mutated metastatic breast cancer. METHODS: The MEDIOLA trial is a multicentre, open-label, phase 1/2, basket trial of durvalumab and olaparib in solid tumours. Patients were enrolled into four initial cohorts: germline BRCA-mutated, metastatic breast cancer; germline BRCA-mutated, metastatic ovarian cancer; metastatic gastric cancer; and relapsed small-cell lung cancer. Here, we report on the cohort of patients with breast cancer. Patients who were aged 18 years or older (or aged 19 years or older in South Korea) with germline BRCA1-mutated or BRCA2-mutated or both and histologically confirmed, progressive, HER2-negative, metastatic breast cancer were enrolled from 14 health centres in the UK, the USA, Israel, France, Switzerland, and South Korea. Patients should not have received more than two previous lines of chemotherapy for metastatic breast cancer. Patients received 300 mg olaparib in tablet form orally twice daily for 4 weeks and thereafter a combination of olaparib 300 mg twice daily and durvalumab 1·5 g via intravenous infusion every 4 weeks until disease progression. Primary endpoints were safety and tolerability, and 12-week disease control rate. Safety was analysed in patients who received at least one dose of study treatment, and activity analyses were done in the full-analysis set (patients who received at least one dose of study treatment and were not excluded from the study). Recruitment has completed and the study is ongoing. This trial is registered with ClinicalTrials.gov, NCT02734004. FINDINGS: Between June 14, 2016, and May 2, 2017, 34 patients were enrolled and received both study drugs and were included in the safety analysis. 11 (32%) patients experienced grade 3 or worse adverse events, of which the most common were anaemia (four [12%]), neutropenia (three [9%]), and pancreatitis (two [6%]). Three (9%) patients discontinued due to adverse events and four (12%) patients experienced a total of six serious adverse events. There were no treatment-related deaths. 24 (80%; 90% CI 64·3-90·9) of 30 patients eligible for activity analysis had disease control at 12 weeks. INTERPRETATION: Combination of olaparib and durvalumab showed promising antitumour activity and safety similar to that previously observed in olaparib and durvalumab monotherapy studies. Further research in a randomised setting is needed to determine predictors of therapeutic benefit and whether addition of durvalumab improves long-term clinical outcomes compared with olaparib monotherapy. FUNDING: AstraZeneca.

Uniform sarcolemmal dystrophin expression is required to prevent extracellular microRNA release and improve dystrophic pathology.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by genetic loss of dystrophin protein. Extracellular microRNAs (ex-miRNAs) are putative, minimally invasive biomarkers of DMD. Specific ex-miRNAs (e.g. miR-1, miR-133a, miR-206, and miR-483) are highly up-regulated in the serum of DMD patients and dystrophic animal models and are restored to wild-type levels following exon skipping-mediated dystrophin rescue in mdx mice. As such, ex-miRNAs are promising pharmacodynamic biomarkers of exon skipping efficacy. Here, we aimed to determine the degree to which ex-miRNA levels reflect the underlying level of dystrophin protein expression in dystrophic muscle. METHODS: Candidate ex-miRNA biomarker levels were investigated in mdx mice in which dystrophin was restored with peptide-PMO (PPMO) exon skipping conjugates and in mdx-XistΔhs mice that express variable amounts of dystrophin from birth as a consequence of skewed X-chromosome inactivation. miRNA profiling was performed in mdx-XistΔhs mice using the FirePlex methodology and key results validated by small RNA TaqMan RT-qPCR. The muscles from each animal model were further characterized by dystrophin western blot and immunofluorescence staining. RESULTS: The restoration of ex-myomiR abundance observed following PPMO treatment was not recapitulated in the high dystrophin-expressing mdx-XistΔhs group, despite these animals expressing similar amounts of total dystrophin protein (~37% of wild-type levels). Instead, ex-miRNAs were present at high levels in mdx-XistΔhs mice regardless of dystrophin expression. PPMO-treated muscles exhibited a uniform pattern of dystrophin localization and were devoid of regenerating fibres, whereas mdx-XistΔhs muscles showed non-homogeneous dystrophin staining and sporadic regenerating foci. CONCLUSIONS: Uniform dystrophin expression is required to prevent ex-miRNA release, stabilize myofiber turnover, and attenuate pathology in dystrophic muscle.

Longitudinal immune characterization of syngeneic tumor models to enable model selection for immune oncology drug discovery.

BACKGROUND: The ability to modulate immune-inhibitory pathways using checkpoint blockade antibodies such as αPD-1, αPD-L1, and αCTLA-4 represents a significant breakthrough in cancer therapy in recent years. This has driven interest in identifying small-molecule-immunotherapy combinations to increase the proportion of responses. Murine syngeneic models, which have a functional immune system, represent an essential tool for pre-clinical evaluation of new immunotherapies. However, immune response varies widely between models and the translational relevance of each model is not fully understood, making selection of an appropriate pre-clinical model for drug target validation challenging. METHODS: Using flow cytometry, O-link protein analysis, RT-PCR, and RNAseq we have characterized kinetic changes in immune-cell populations over the course of tumor development in commonly used syngeneic models. RESULTS: This longitudinal profiling of syngeneic models enables pharmacodynamic time point selection within each model, dependent on the immune population of interest. Additionally, we have characterized the changes in immune populations in each of these models after treatment with the combination of α-PD-L1 and α-CTLA-4 antibodies, enabling benchmarking to known immune modulating treatments within each model. CONCLUSIONS: Taken together, this dataset will provide a framework for characterization and enable the selection of the optimal models for immunotherapy combinations and generate potential biomarkers for clinical evaluation in identifying responders and non-responders to immunotherapy combinations.

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 ~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.

PI3Kα/δ inhibition promotes anti-tumor immunity through direct enhancement of effector CD8+ T-cell activity.

PI3K inhibitors with differential selectivity to distinct PI3K isoforms have been tested extensively in clinical trials, largely to target tumor epithelial cells. PI3K signaling also regulates the immune system and inhibition of PI3Kδ modulate the tumor immune microenvironment of pre-clinical mouse tumor models by relieving T-regs-mediated immunosuppression. PI3K inhibitors as a class and PI3Kδ specifically are associated with immune-related side effects. However, the impact of mixed PI3K inhibitors in tumor immunology is under-explored. Here we examine the differential effects of AZD8835, a dual PI3Kα/δ inhibitor, specifically on the tumor immune microenvironment using syngeneic models. Continuous suppression of PI3Kα/δ was not required for anti-tumor activity, as tumor growth inhibition was potentiated by an intermittent dosing/schedule in vivo. Moreover, PI3Kα/δ inhibition delivered strong single agent anti-tumor activity, which was associated with dynamic suppression of T-regs, improved CD8+ T-cell activation and memory in mouse syngeneic tumor models. Strikingly, AZD8835 promoted robust CD8+ T-cell activation dissociated from its effect on T-regs. This was associated with enhancing effector cell viability/function. Together these data reveal novel mechanisms by which PI3Kα/δ inhibitors interact with the immune system and validate the clinical compound AZD8835 as a novel immunoncology drug, independent of effects on tumor cells. These data support further clinical investigation of PI3K pathway inhibitors as immuno-oncology agents.

Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD.

Extracellular small RNAs (sRNAs), including microRNAs (miRNAs), are promising biomarkers for diseases such as Duchenne muscular dystrophy (DMD), although their biological relevance is largely unknown. To investigate the relationship between intracellular and extracellular sRNA levels on a global scale, we performed sRNA sequencing in four muscle types and serum from wild-type, dystrophic mdx, and mdx mice in which dystrophin protein expression was restored by exon skipping. Differentially abundant sRNAs were identified in serum (mapping to miRNA, small nuclear RNA [snRNA], and PIWI-interacting RNA [piRNA] loci). One novel candidate biomarker, miR-483, was increased in both mdx serum and muscle, and also elevated in DMD patient sera. Dystrophin restoration induced global shifts in miRNA (including miR-483) and snRNA-fragment abundance toward wild-type levels. Specific serum piRNA-like sRNAs also responded to exon skipping therapy. Absolute miRNA expression in muscle was positively correlated with abundance in the circulation, although multiple highly expressed miRNAs in muscle were not elevated in mdx serum, suggesting that both passive and selective release mechanisms contribute to serum miRNA levels. In conclusion, this study has revealed new insights into the sRNA biology of dystrophin deficiency and identified novel DMD biomarkers.

Evaluation of methodologies for microRNA biomarker detection by next generation sequencing.

In recent years, microRNAs (miRNAs) in tissues and biofluids have emerged as a new class of promising biomarkers for numerous diseases. Blood-based biomarkers are particularly desirable since serum or plasma is easily accessible and can be sampled repeatedly. To comprehensively explore the biomarker potential of miRNAs, sensitive, accurate and cost-efficient miRNA profiling techniques are required. Next generation sequencing (NGS) is emerging as the preferred method for miRNA profiling; offering high sensitivity, single-nucleotide resolution and the possibility to profile a considerable number of samples in parallel. Despite the excitement about miRNA biomarkers, challenges associated with insufficient characterization of the sequencing library preparation efficacy, precision and method-related quantification bias have not been addressed in detail and are generally underappreciated in the wider research community. Here, we have tested in parallel four commercially available small RNA sequencing kits against a cohort of samples comprised of human plasma, human serum, murine brain tissue and a reference library containing ~ 950 synthetic miRNAs. We discuss the advantages and limits of these methodologies for massive parallel microRNAs profiling. This work can serve as guideline for choosing an adequate library preparation method, based on sensitivity, specificity and accuracy of miRNA quantification, workflow convenience and potential for automation.

Biomarker Potential of Extracellular miRNAs in Duchenne Muscular Dystrophy.

miRNAs are small, noncoding RNAs that not only regulate gene expression within cells, but might also constitute promising extracellular biomarkers for a variety of pathologies, including the progressive muscle-wasting disorder Duchenne Muscular Dystrophy (DMD). A set of muscle-enriched miRNAs, the myomiRs (miR-1, miR-133, and miR-206) are highly elevated in the serum of patients with DMD and in dystrophin-deficient animal models. Furthermore, circulating myomiRs might be used as pharmacodynamic biomarkers, given that their levels can be restored towards wild-type levels following exon skipping therapy in dystrophic mice. The relationship between muscle pathology and extracellular myomiR release is complex, and incompletely understood. Here, we discuss current progress leading towards the clinical utility of extracellular miRNAs as putative DMD biomarkers, and their possible contribution to muscle physiology.

Selective release of muscle-specific, extracellular microRNAs during myogenic differentiation.

MyomiRs are muscle-specific microRNAs (miRNAs) that regulate myoblast proliferation and differentiation. Extracellular myomiRs (ex-myomiRs) are highly enriched in the serum of Duchenne Muscular Dystrophy (DMD) patients and dystrophic mouse models and consequently have potential as disease biomarkers. The biological significance of miRNAs present in the extracellular space is not currently well understood. Here we demonstrate that ex-myomiR levels are elevated in perinatal muscle development, during the regenerative phase that follows exercise-induced myoinjury, and concomitant with myoblast differentiation in culture. Whereas ex-myomiRs are progressively and specifically released by differentiating human primary myoblasts and C2C12 cultures, chemical induction of apoptosis in C2C12 cells results in indiscriminate miRNA release. The selective release of myomiRs as a consequence of cellular differentiation argues against the idea that they are solely waste products of muscle breakdown, and suggests they may serve a biological function in specific physiological contexts. Ex-myomiRs in culture supernatant and serum are predominantly non-vesicular, and their release is independent of ceramide-mediated vesicle secretion. Furthermore, ex-myomiRs levels are reduced in aged dystrophic mice, likely as a consequence of chronic muscle wasting. In conclusion, we show that myomiR release accompanies periods of myogenic differentiation in cell culture and in vivo. Serum myomiR abundance is therefore a function of the regenerative/degenerative status of the muscle, overall muscle mass, and tissue expression levels. These findings have implications for the use of ex-myomiRs as biomarkers for DMD disease progression and monitoring response to therapy.

Identification of novel, therapy-responsive protein biomarkers in a mouse model of Duchenne muscular dystrophy by aptamer-based serum proteomics.

There is currently an urgent need for biomarkers that can be used to monitor the efficacy of experimental therapies for Duchenne Muscular Dystrophy (DMD) in clinical trials. Identification of novel protein biomarkers has been limited due to the massive complexity of the serum proteome and the presence of a small number of very highly abundant proteins. Here we have utilised an aptamer-based proteomics approach to profile 1,129 proteins in the serum of wild-type and mdx (dystrophin deficient) mice. The serum levels of 96 proteins were found to be significantly altered (P < 0.001, q < 0.01) in mdx mice. Additionally, systemic treatment with a peptide-antisense oligonucleotide conjugate designed to induce Dmd exon skipping and recover dystrophin protein expression caused many of the differentially abundant serum proteins to be restored towards wild-type levels. Results for five leading candidate protein biomarkers (Pgam1, Tnni3, Camk2b, Cycs and Adamts5) were validated by ELISA in the mouse samples. Furthermore, ADAMTS5 was found to be significantly elevated in human DMD patient serum. This study has identified multiple novel, therapy-responsive protein biomarkers in the serum of the mdx mouse with potential utility in DMD patients.