Journal: Brain

Article Title: Engineered microRNA-based regulatory element permits safe high-dose mini MECP2 gene therapy in Rett mice

doi: 10.1093/brain/awab182

Figure Lengend Snippet: Schematic of strategy used to rationally synthesize a miRNA target panel tailored for Rett syndrome. (A) The first five steps of a conceptual negative feedback loop are indicated: (1) viral genome in nucleus; (2) exogenous MECP2-myc mRNA in cytoplasm; (3) translation and nuclear localization of MECP2-myc; (4) exogenous MECP2-myc upregulates miRNA expression; (5) mature miRNAs bind to non-coding targets in the 3ʹ UTR of an exogenous MECP2-myc transcript. After mature miRNAs bind to targets in the exogenous MECP2 mRNA, endogenous RNAi machinery (not shown) may conditionally silence exogenous MECP2 expression. The targets inserted into the viral genome may match targets found in the endogenous Mecp2 mRNA. Conceptually, this model could be adapted for other potentially toxic transcription factors that drive the expression of miRNAs in vivo. Alternatively, insertion of miRNA targets into the viral genome could improve the safety of gene therapies encoding potentially toxic cytoplasmic proteins, provided that those proteins indirectly upregulate miRNAs. (B) To identify translationally relevant miRNAs upregulated by toxic MECP2 gene transfer, (1) adolescent wild-type (WT) and knockout (KO) mice were treated intracisternally with saline, 1 × 1012 vg AAV9/MECP2, or 1 × 1012 vg AAV9/EGFP. (2) Two to three weeks later, CNS tissue nearest to the injection site (cerebellum, medulla and cervical cord; see dashed circle) was harvested from treated mice. The image shown in step 2 illustrates the spread of dye after ICM administration. The relatively concentrated dye localization near the cerebellum indicates how vulnerable tissue near the intracisternal site may be after treatment with high titre, toxic virus. (3) RNA was purified from dissected tissue, frozen and shipped to LC Sciences for miRNA profiling. Data from this screen were subsequently used to rank candidate miRNA targets from a secondary bioinformatics approach.

Article Snippet: MiRNA profiling LC Sciences profiled mouse miRNAs from blinded RNA samples and conducted statistical analyses (microarray part number MRA-1002; miRBase version 21; n = three mice per group per tissue type with two screening replicates).

Techniques: Expressing, In Vivo, Knock-Out, Saline, Injection, Virus, Purification

Journal: Brain

Article Title: Engineered microRNA-based regulatory element permits safe high-dose mini MECP2 gene therapy in Rett mice

doi: 10.1093/brain/awab182

Figure Lengend Snippet: Putative positive hits upregulated in correlation with endogenous or exogenous MECP2 according to miRNA microarray. (A–F) Significant differences between saline-treated knockout (KO) and saline-treated wild-type (WT) mice. (G–L) Significant differences between saline-treated and AAV9/MECP2-treated mice. (A–L) None of the 12 miRNAs listed here were significantly upregulated by AAV9/EGFP (versus saline-treated knockout or wild-type mice; P > 0.05). Each data-point represents the average of two screening replicates; error bars for each mean are not shown. n = 3 mice per group. P < 0.05 between the groups boxed in red. *A ‘reg1’ miRNA target panel featuring binding sites for miR-451a, miR-690, and let-7e-5p failed to improve the safety of miniMECP2 gene transfer (data not shown). As discussed in the main text, negative results (P > 0.05) should be interpreted with caution, as miRNAs upregulated at the cell-type level may be masked by noise from other cell types in the same tissue. Furthermore, low normalized fluorescence intensities may reflect low miRNA expression levels in a tissue sample but may fail to reveal high expression levels within a single cell type within said tissue. In general, many of the significant differences in normalized fluorescence intensity are for relatively small increases in mean signal between the indicated treatment groups. These subtle changes, along with the general noisiness of the data, warranted a secondary technique to justify the use of specific miRNA targets within a new target panel design. Finally, miRNAs significantly upregulated by AAV9/EGFP (versus saline-treated knockout mice) are miR-99a-5p, miR-107-3p and let-7f-5p in cervical cord (CC; data not shown). MiRNAs upregulated by AAV9/EGFP (versus saline-treated wild-type mice) are miR-669a-3p in cervical cord; let-7j in cerebellum; and miR-669f-3p, miR-669p-3p, miR-30c-5p and miR-669a-3p in medulla (data not shown).

Article Snippet: MiRNA profiling LC Sciences profiled mouse miRNAs from blinded RNA samples and conducted statistical analyses (microarray part number MRA-1002; miRBase version 21; n = three mice per group per tissue type with two screening replicates).

Techniques: Microarray, Saline, Knock-Out, Binding Assay, Fluorescence, Expressing

Journal: Brain

Article Title: Engineered microRNA-based regulatory element permits safe high-dose mini MECP2 gene therapy in Rett mice

doi: 10.1093/brain/awab182

Figure Lengend Snippet: Microarray expression data were used to rank targets that appear frequently across a curated list of 3ʹ UTRs for genes mediating intellectual disability. (A) Many miRNA targets appear frequently across the curated list of 3ʹ UTRs. Among 2491 human targets and 1831 mouse targets, 451 targets had identical annotation across both mouse and human 3ʹ UTR datasets. (B) The scatter plot shows the same data as those shown in A. Targets that are annotated in over half of the examined 3ʹ UTR sequences for both species were prioritized for target panel design (shaded area). Within the shaded area, purple data-points indicate targets corresponding to miRNAs expressed at moderate to high levels in dissected cervical cord, cerebellar, and/or medullar tissue (signal intensity >500). Square data-points indicate targets for putative MECP2-responsive miRNAs indicated in Fig. 3A, C, D and G–J as well as two additional miRNAs (miR-9-5p and miR-27a-3p; indicated by a hash symbol), which showed trends in increased expression in cervical cord when data for MECP2− and MECP2+ treatment groups were aggregated together (data not shown). MECP2− groups, for example, would include both saline-treated and AAV9/EGFP-treated knockout mice. For clarity, we have limited notation (purple and square data-points) to the shaded area only. The double dagger symbol (‡) indicates the target for miR-218-5p included in the new panel design (‘miRARE’) for the purpose of making miRARE more broadly applicable for multiple gene therapy applications. Expression of miR-218-5p did not appear to be MECP2-responsive in our high throughput screening data. (C) Cartoon of the miniMECP2-miRARE viral genome cassette. Yellow and green lines indicate miRNA targets that are part of the previously published RDH1pA.4 Blue lines indicate miRARE targets that were inserted into RDH1pA. ITR = inverted terminal repeat; sc = mutated self-complementary ITR sequence.

Article Snippet: MiRNA profiling LC Sciences profiled mouse miRNAs from blinded RNA samples and conducted statistical analyses (microarray part number MRA-1002; miRBase version 21; n = three mice per group per tissue type with two screening replicates).

Techniques: Microarray, Expressing, Saline, Knock-Out, High Throughput Screening Assay, Sequencing

Journal: PLoS ONE

Article Title: Differential methylation and expression patterns of microRNAs in relation to breast cancer subtypes among American women of African and European ancestry

doi: 10.1371/journal.pone.0249229

Figure Lengend Snippet: (A) The heatmap shows the Spearman’s correlation coefficients (rho) between each CpG-miR pair ( P <0.05) in AA and EA tumors. Red bars represent positive correlation and blue bars represent negative correlation. (B) The Scatter plots relate the methylation and expression levels on selected top correlated CpG-miR pairs including specific probes with miR-190b-5p, miR-135b-5p, miR-224-5p, and miR-452-3p. For each CpG-miR pair, the methylation level (beta value) is on the X axis, and the expression level (log counts per million, logCPM) of corresponding miRNA is on Y axis. The Spearman’s correlation coefficient (rho) of each CpG-miR pair for AA and EA tumors was presented in the figure.

Article Snippet: To validate our findings, TCGA DNA methylation and miRNA expression data were downloaded from GDAC Broad Institute ( http://gdac.broadinstitute.org/runs/stddata 2016_01_28/data/BRCA/20160128/).

Techniques: Methylation, Expressing

Journal: Frontiers in Allergy

Article Title: miR-155 Is a Positive Regulator of FcεRI-Induced Cyclooxygenase-2 Expression and Cytokine Production in Mast Cells

doi: 10.3389/falgy.2022.835776

Figure Lengend Snippet: miRNA profiling in human SMCs. Heat map showing significant ( p < 0.01) changes in miRNA expression in human SMCs following FcεRI crosslinking. RNA was extracted from SMCs that were sensitized with chimeric human anti-NP IgE (1 μg/ml) and activated for 3 h at 37°C with the multivalent antigen NP-HSA (1 ng/ml). The heat map identifies 10 miRNAs that were significantly ( p < 0.01) upregulated (red) and 11 that were significantly downregulated (green) in Ag/IgE-activated SMCs ( n = 3 different cultures from different donors). All data can be found in the .

Article Snippet: miRNA profiling services were performed by LC Sciences (lcsciences.com, Houston TX, USA) using their μParaflo ® microfluidic biochip technology and optimized probes in a microfluidics microarray platform.

Techniques: Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Clinical Significance of PTEN Deletion, Mutation, and Loss of PTEN Expression in De Novo Diffuse Large B-Cell Lymphoma

doi: 10.1016/j.neo.2018.03.002

Figure Lengend Snippet: miRNA profiling and gene expression profiling analysis in the training cohort. (A-B) Loss of cytoplasmic PTEN expression was associated with significantly higher levels of miR-106b-3p, miR-200c-5p, miR-486-5p, miR-141-5p, and miR-130b-5p expression in DLBCL. (C) Absence of PD-L1 expression was associated with significantly higher miR-106b-3p expression. (D) Low PTEN mRNA expression was associated with significantly worse progression-free survival (PFS) in GCB-DLBCL, ABC-DLBCL, and the p-AKT high DLBCL subset. (E) Genes significantly differently expressed between DLBCL groups with low PTEN mRNA expression and other cases (designated as PTEN low and PTEN not low , respectively), and between DLBCL patients with wild-type PTEN ( WT-PTEN ) and mutated PTEN ( MUT-PTEN ).

Article Snippet: Multiple t -tests were used to identify differentially expressed genes between groups with and without PTEN abnormalities, and the P values obtained were corrected for the false discovery rate (FDR) using the beta-uniform mixture method. microRNA (miRNA) profiling was performed by HTG Molecular Diagnostics Inc. (Tucson, AZ) using FFPE tissue sections (unpublished preliminary data). miRNAs targeting PTEN are according to the literature review and TargetScan: http://www.targetscan.org ).

Techniques: Gene Expression, Expressing