Xavier Bofill-De Ros

MiR-148a- and miR-216a-regulated Oncolytic Adenoviruses Targeting Pancreatic Tumors Attenuate Tissue Damage Without Perturbation of miRNA Activity

Oncolytic virotherapy shows promise for pancreatic ductal adenocarcinoma (PDAC) treatment, but there is the need to minimize associated-toxicities. In the current work, we engineered artificial target sites recognized by miR-216a and/or miR-148a to provide pancreatic tumor-selectivity to replication-competent adenoviruses (Ad-miRTs) and improve their safety profile. Expression analysis in PDAC patients identified miR-148a and miR-216a downregulated in resectable (FC(miR-148a) = 0.044, P < 0.05; FC(miR-216a) = 0.017, P < 0.05), locally advanced (FC(miR-148a) = 0.038, P < 0.001; FC(miR-216a) = 0.001, P < 0.001) and metastatic tumors (FC(miR-148a) = 0.041, P < 0.01; FC(miR-216a) = 0.002, P < 0.001). In mouse tissues, miR-216a was highly specific of the exocrine pancreas whereas miR-148a was abundant in the exocrine pancreas, Langerhans islets, and the liver. In line with the miRNA content and the miRNA target site design, we show E1A gene expression and viral propagation efficiently controlled in Ad-miRT-infected cells. Consequently, Ad-miRT-infected mice presented reduced pancreatic and liver damage without perturbation of the endogenous miRNAs and their targets. Interestingly, the 8-miR148aT design showed repressing activity by all miR-148/152 family members with significant detargeting effects in the pancreas and liver. Ad-miRTs preserved their oncolytic activity and triggered strong antitumoral responses. This study provides preclinical evidences of miR-148a and miR-216a target site insertions to confer adenoviral selectivity and proposes 8-miR148aT as an optimal detargeting strategy for genetically-engineered therapies against PDAC.

Genome-wide miR-155 and miR-802 target gene identification in the hippocampus of Ts65Dn Down syndrome mouse model by miRNA sponges

BackgroundDown syndrome (DS) or trisomy 21 is the result of a genetic dosage imbalance that translates in a broad clinical spectrum. A major challenge in the study of DS is the identification of functional genetic elements with wide impact on phenotypic alterations. Recently, miRNAs have been recognized as major contributors to several disease conditions by acting as post-transcriptional regulators of a plethora of genes. Five chromosome 21 (HSA21) miRNAs have been found overexpressed in DS individuals and could function as key elements in the pathophysiology. Interestingly, in the trisomic Ts65Dn DS mouse model two of these miRNAs (miR-155 and miR-802) are also triplicated and overexpressed in brain.ResultsIn the current work, we interrogated the impact of miR-155 and miR-802 upregulation on the transcriptome of Ts65Dn brains. We developed a lentiviral miRNA-sponge strategy (Lv-miR155-802T) to identify in vivo relevant miR-155 and miR-802 target mRNAs. Hippocampal injections of lentiviral sponges in Ts65Dn mice normalized the expression of miR-155 and miR-802 and rescued the levels of their targets methyl-CpG-binding protein 2 gene (Mecp2), SH2 (Src homology 2)-containing inositol phosphatase-1 (Ship1) and Forkhead box protein M1 (FoxM1). Transcriptomic data of Lv-miR155-802T miRNA-sponge treated hippocampi correlated with candidate targets highlighting miRNA dosage-sensitive genes. Significant associations were found in a subset of genes (Rufy2, Nova1, Nav1, Thoc1 and Sumo3) that could be experimentally validated.ConclusionsThe lentiviral miRNA-sponge strategy demonstrated the genome-wide regulatory effects of miR-155 and miR-802. Furthermore, the analysis combining predicted candidates and experimental transcriptomic data proved to retrieve genes with potential significance in DS-hippocampal phenotype bridging with DS other neurological-associated diseases such as Alzheimer’s disease.

Guidelines for the optimal design of miRNA-based shRNAs.

RNA interference (RNAi) is an extremely useful tool for inhibiting gene expression. It can be triggered by transfected synthetic small interfering RNA (siRNA) or by expressed small hairpin RNA (shRNA). The cellular machinery processes the latter into siRNA in vivo. shRNA is preferred or required in genetic screens and specific RNAi approaches in gene therapy settings. Despite its many successes, the field of shRNAs faces many challenges. Insufficient knockdowns and off-target effects become obstacles for shRNA usage in many applications. Numerous failures are triggered by pitfalls in shRNA design that is often associated with impoverished biogenesis. Here, based on current understanding of the miRNA maturation pathway, we discuss the principles of different shRNA design (pre-miRNA-like, pri-miRNA-like and Ago-shRNA) with an emphasis on the RNA structure. We also provide detailed instructions for an optimal design of pre-miRNA-like shRNA.

Stress-Induced MicroRNA-708 Impairs β-Cell Function and Growth

The pancreatic β-cell transcriptome is highly sensitive to external signals such as glucose oscillations and stress cues. MicroRNAs (miRNAs) have emerged as key factors in gene expression regulation. Here, we aimed to identify miRNAs that are modulated by glucose in mouse pancreatic islets. We identified miR-708 as the most upregulated miRNA in islets cultured at low glucose concentrations, a setting that triggers a strong stress response. miR-708 was also potently upregulated by triggering endoplasmic reticulum (ER) stress with thapsigargin and in islets of ob/ob mice. Low-glucose induction of miR-708 was blocked by treatment with the chemical chaperone 4-phenylbutyrate, uncovering the involvement of ER stress in this response. An integrative analysis identified neuronatin (Nnat) as a potential glucose-regulated target of miR-708. Indeed, Nnat expression was inversely correlated with miR-708 in islets cultured at different glucose concentrations and in ob/ob mouse islets and was reduced after miR-708 overexpression. Consistent with the role of Nnat in the secretory function of β-cells, miR-708 overexpression impaired glucose-stimulated insulin secretion (GSIS), which was recovered by NNAT overexpression. Moreover, miR-708 inhibition recovered GSIS in islets cultured at low glucose. Finally, miR-708 overexpression suppressed β-cell proliferation and induced β-cell apoptosis. Collectively, our results provide a novel mechanism of glucose regulation of β-cell function and growth by repressing stress-induced miR-708.

Controlling Adenoviral Replication to Induce Oncolytic Efficacy

Over the last decade, cancer therapy has found itself challenged by the growing field of oncolytic virotherapy. Many different viruses are currently under study, investigating their potential to induce antitumor effects through repeated cycles of viral infection and cell lysis. It was, however, genetically-engineered replication-selective adenoviruses that were the first to enter clinical trials with cancer patients. The difficulties involved in combining selectivity and elevated potency in a single oncolytic adenovirus have led investigators to design and test many different approaches. Different strategies, based on the control of viral replication, are presented in the current review. We discuss how the growing knowledge of cell and tumour biology, with the advances made in adenoviral virology, has inspired the fine-tuning of genetically-engineered adenoviruses. Special emphasis is placed on the fundamentals behind the use of certain specific genetic elements, introduced into the viral genome to control viral gene expression and on describing the most important viral gene mutations.

Implications of MicroRNAs in Oncolytic Virotherapy

MicroRNAs (miRNAs) are an abundant class of small non-coding RNA molecules (~22u2009nt) that can repress gene expression. Deregulation of certain miRNAs is widely recognized as a robust biomarker for many neoplasms, as well as an important player in tumorigenesis and the establishment of tumoral microenvironments. The downregulation of specific miRNAs in tumors has been exploited as a mechanism to provide selectivity to oncolytic viruses or gene-based therapies. miRNA response elements recognizing miRNAs expressed in specific tissues, but downregulated in tumors, have been inserted into the 3′UTR of viral genes to promote the degradation of these viral mRNAs in healthy tissue, but not in tumor cells. Consequently, oncolytic virotherapy-associated toxicities were diminished, while therapeutic activity in tumor cells was preserved. However, viral infections themselves can modulate the miRNome of the host cell, and such miRNA changes under infection impact the normal viral lifecycle. Thus, there is a miRNA-mediated interplay between virus and host cell, affecting both viral and cellular activities. Moreover, the outcome of such interactions may be cell type or condition specific, suggesting that the impact on normal and tumoral cells may differ. Here, we provide an insight into the latest developments in miRNA-based viral engineering for cancer therapy, following the most recent discoveries in miRNA biology. Furthermore, we report on the relevance of miRNAs in virus–host cell interaction, and how such knowledge can be exploited to improve the control of viral activity in tumor cells.

Structural differences between pri-miRNA paralogs promotes alternative Drosha cleavage and expands target repertoires

MicroRNA (miRNA) processing begins with Drosha cleavage, the fidelity of which is critical for downstream processing and mature miRNA target specificity. To understand how pri-miRNA sequence and structure influence Drosha cleavage, we studied the maturation of three pri-miR-9 paralogs, which encode the same mature miRNA but differ in the surrounding scaffold. We show that pri-miR-9-1 has a unique Drosha cleavage profile due to its distorted and flexible stem structure. Cleavage of pri-miR-9-1, but not pri-miR-9-2 or pri-miR-9-3, generates an alternative-miR-9 with a shifted seed sequence that expands the scope of its target RNAs. Analyses of low grade glioma patient samples indicate that the alternative-miR-9 plays a distinct role in preventing tumor progression. To generalize our model, we provide evidence that distortion of pri-miRNA stems correlates with Drosha cleavage at non-canonical sites. Our studies reveal that pri-miRNA paralogs can have distinct functions via differential Drosha processing.

DYRK1A modulates c-MET in pancreatic ductal adenocarcinoma to drive tumour growth

Background and aims Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumour with a poor prognosis using current treatments. Targeted therapies may offer a new avenue for more effective strategies. Dual-specificity tyrosine regulated kinase 1A (DYRK1A) is a pleiotropic kinase with contradictory roles in different tumours that is uncharacterised in PDAC. Here, we aimed to investigate the role of DYRK1A in pancreatic tumorigenesis. Design We analysed DYRK1A expression in PDAC genetic mouse models and in patient samples. DYRK1A function was assessed with knockdown experiments in pancreatic tumour cell lines and in PDAC mouse models with genetic reduction of Dyrk1a dosage. Furthermore, we explored a mechanistic model for DYRK1A activity. Results We showed that DYRK1A was highly expressed in PDAC, and that its protein level positively correlated with that of c-MET. Inhibition of DYRK1A reduced tumour progression by limiting tumour cell proliferation. DYRK1A stabilised the c-MET receptor through SPRY2, leading to prolonged activation of extracellular signal-regulated kinase signalling. Conclusions These findings reveal that DYRK1A contributes to tumour growth in PDAC, at least through regulation of c-MET accumulation, suggesting that inhibition of DYRK1A could represent a novel therapeutic target for PDAC.

QuagmiR: A Cloud-based Application for IsomiR Big Data Analytics

SUMMARYnMicroRNAs (miRNAs) function as master regulators of gene expression. Recent studies demonstrate that miRNA isoforms (isomiRs) play a unique role in cancer development. Here, we present QuagmiR, the first cloud-based tool to analyze isomiRs from next generation sequencing data. Using a novel and flexible searching algorithm designed for the detection and annotation of heterogeneous isomiRs, it permits extensive customization of the query process and reference databases to meet the useru2009s diverse research needs.nnnAVAILABILITY AND IMPLEMENTATIONnQuagmiR is written in Python and can be obtained freely from GitHub (https://github.com/Gu-Lab-RBL-NCI/QuagmiR). QuagmiR can be run from the command line on local machines, as well as on high-performance servers. A web-accessible version of the tool has also been made available for use by academic researchers through the National Cancer Institute-funded Seven Bridges Cancer Genomics Cloud (https://cancergenomicscloud.org).nnnSUPPLEMENTARY INFORMATIONnSupplementary data are available at Bioinformatics online.

154. Applying Insights from Pri-miRNA Processing to shRNA Design

Short hairpin RNA (shRNA) technology permits efficient and stable gene regulation, providing a useful tool for gene therapy. shRNAs can be embedded in miRNA scaffolds that allow their expression from Pol II promoters, which is more amendable to control transcription. Under this platform, shRNAs are processed through the canonical miRNA maturation pathway, which includes stepwise cleavages by Drosha-DGCR8 complex (Microprocessor) and Dicer. Despite many successes, design of Pol II driven shRNA is far from optimal. Most Pol II driven shRNAs are based on primary transcript sequence of hsa-miR-30a. Recent studies have shown that different pri-miRNAs have distinct Drosha cleavage efficiencies leading to various levels of its mature form. Here, we seek to systematically explore how different pri-miRNA scaffolds can be adapted and optimized to provide the best shRNA expression and function. By taking advantage of DGCR8 KO cells and luciferase-based reporters, we were able to directly monitor Microprocessor cleavage efficiency of primary transcripts in vivo. We have analyzed the Microprocessor cleavage of 20 different primary miRNA transcripts that are the most abundant and widely expressed. Moreover, we have explored how the preservation of stem structure and endogenous surrounding sequences and motifs contribute to shRNA maturation. This study aims to pave the way for a universal miRNA-based shRNA expression platform.