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The adoption of whole genome sequencing in genetic screens has facilitated the detection of genetic variation in the intronic regions of genes, far from annotated splice sites. However, selecting an appropriate computational tool to differentiate functionally relevant genetic variants from those with no effect is challenging, particularly for deep intronic regions where independent benchmarks are scarce.In this study, we have provided an overview of the computational methods available and the extent to which they can be used to analyze deep intronic variation. We leveraged diverse datasets to extensively evaluate tool performance across different intronic regions, distinguishing between variants that are expected to disrupt splicing through different molecular mechanisms. Notably, we compared the performance of SpliceAI, a widely used sequence-based deep learning model, with that of more recent methods that extend its original implementation. We observed considerable differences in tool performance depending on the region considered, with variants generating cryptic splice sites being better predicted than those that affect splicing regulatory elements or the branchpoint region. Finally, we devised a novel quantitative assessment of tool interpretability and found that tools providing mechanistic explanations of their predictions are often correct with respect to the ground truth information, but the use of these tools results in decreased predictive power when compared to black box methods.Our findings translate into practical recommendations for tool usage and provide a reference framework for applying prediction tools in deep intronic regions, enabling more informed decision-making by practitioners.

© Translational Lung Cancer Research. All rights reserved. This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/., Background: Liquid biopsy allows the identification of targetable cancer mutations in a minimally invasive manner. In patients with advanced non-small cell lung cancer (NSCLC), droplet digital PCR (ddPCR) is increasingly used to genotype the epidermal growth factor receptor (EGFR) gene in circulating cell-free DNA (cfDNA). However, the sensitivity of this method is still under debate. The aim of this study was to implement and assess the performance of a ddPCR assay for detecting the EGFR T790M mutation in liquid biopsies. Methods: A ddPCR assay was optimized to detect the EGFR T790M mutation in plasma samples from 77 patients with NSCLC in progression. Results: Our ddPCR assay enabled the detection and quantification of the EGFR T790M mutation at cfDNA allele frequency as low as 0.5%. The mutation was detected in 40 plasma samples, corresponding to a positivity rate of 52%. The number of mutant molecules per mL of plasma ranged from 1 to 6,000. A re-biopsy was analyzed for 12 patients that had a negative plasma test and the mutation was detected in 2 cases. A second liquid biopsy was performed for 6 patients and the mutation was detected in 3 cases. Conclusions: This study highlights the value of ddPCR to detect and quantify the EGFR T790M mutation in liquid biopsies in a real-world clinical setting. Our results suggest that repeated ddPCR tests in cfDNA may obviate tissue re-biopsy in patients unable to provide a tumor tissue sample suitable for molecular analysis., This work was supported by Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior - Fundos do Orçamento de Estado (UID/BIM/50005/2019), and FCT/FEDER/POR Lisboa 2020, Programa Operacional Regional de Lisboa, PORTUGAL 2020 (LISBOA-01-0145-FEDER-016394). C.S. was a recipient of a FCT fellowship (SFRH/BDE/110544/2015).

Although mutation profiling of defined genes is recommended for classification of acute myeloid leukemia (AML) patients, screening of targeted gene panels using next-generation sequencing (NGS) is not always routinely used as standard of care. The objective of this study was to prospectively assess whether extended molecular monitoring using NGS adds clinical value for risk assessment in real-world AML patients. We analyzed a cohort of 268 newly diagnosed AML patients. We compared the prognostic stratification of our study population according to the European LeukemiaNet recommendations, before and after the incorporation of the extended mutational profile information obtained by NGS. Without access to NGS data, 63 patients (23%) failed to be stratified into risk groups. After NGS data, only 27 patients (10%) failed risk stratification. Another 33 patients were re-classified as adverse-risk patients once the NGS data was incorporated. In total, access to NGS data refined risk assessment for 62 patients (23%). We further compared clinical outcomes with prognostic stratification, and observed unexpected outcomes associated with FLT3 mutations. In conclusion, this study demonstrates the prognostic utility of screening AML patients for multiple gene mutations by NGS and underscores the need for further studies to refine the current risk classification criteria. info:eu-repo/semantics/publishedVersion

Niemann-Pick type A (NPA) disease is a fatal lysosomal neurodegenerative disorder caused by the deficiency in acid sphingomyelinase (ASM) activity. NPA patients present severe and progressive neurodegeneration starting at an early age. Currently, there is no effective treatment for this disease and NPA patients die between 2 and 3 years of age. NPA is characterized by an accumulation of sphingomyelin in lysosomes and dysfunction in the autophagy-lysosomal pathway. Recent studies show that c-Abl tyrosine kinase activity downregulates autophagy and the lysosomal pathway. Interestingly, this kinase is also activated in other lysosomal neurodegenerative disorders. Here, we describe that c-Abl activation contributes to the mechanisms of neuronal damage and death in NPA disease. Our data demonstrate that: 1) c-Abl is activated in-vitro as well as in-vivo NPA models; 2) imatinib, a clinical c-Abl inhibitor, reduces autophagy-lysosomal pathway alterations, restores autophagy flux, and lowers sphingomyelin accumulation in NPA patient fibroblasts and NPA neuronal models and 3) chronic treatment with nilotinib and neurotinib, two c-Abl inhibitors with differences in blood-brain barrier penetrance and target binding mode, show further benefits. While nilotinib treatment reduces neuronal death in the cerebellum and improves locomotor functions, neurotinib decreases glial activation, neuronal disorganization, and loss in hippocampus and cortex, as well as the cognitive decline of NPA mice. Our results support the participation of c-Abl signaling in NPA neurodegeneration and autophagy-lysosomal alterations, supporting the potential use of c-Abl inhibitors for the clinical treatment of NPA patients.

Predictive biomarkers are crucial in clarifying the best strategy to use poly(ADP-ribose) polymerase inhibitors (PARPi) for the greatest benefit to ovarian cancer patients. PARPi are specifically lethal to cancer cells that cannot repair DNA damage by homologous recombination (HR), and HR deficiency is frequently associated with

Objectives Mutations in the gene that encodes epidermal growth factor receptor (EGFR) are biomarkers that predict how non-small cell lung cancer (NSCLC) patients respond to EGFR-targeted therapies collectively known as tyrosine kinase inhibitors (TKIs). Thus, EGFR genotyping provides crucial information for treatment decision. Both Sanger sequencing and real-time PCR methodologies are used for EGFR genotyping. However, methods based on real-time PCR have limitations, as they may not detect rare or novel mutations. The aim of this study was to determine the prevalence of rare mutations in the tyrosine kinase domain (exons 18–21) of the EGFR gene not targeted by the most frequently used real-time PCR approaches, i.e., the cobas® EGFR Mutation Test, and the Idylla™ EGFR Mutation Assay. Methods A total of 1228 NSCLC patients were screened for mutations in exons 18–21 of the EGFR gene using Sanger sequencing. Results We observed that 252 patients (∼20%) had at least one mutation in the EGFR gene, and 38 (∼3%) carried uncommon genetic alterations that would not be identified by the cobas® or the Idylla™ tests. We further found six new single mutations and seven previously unreported compound mutations. Clinical information and patient outcome are presented for these cases. Conclusions This study highlights the value of sequencing-based approaches to identify rare mutations. Our results add to the inventory of known EGFR mutations, thus contributing to improved lung cancer precision treatment.

Widespread cotranscriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either

Women who inherit heterozygous mutations in the BRCA2 gene have an increased risk of developing cancer, mainly breast and ovarian tumors. A particular BRCA2 mutation (c.156_157insAlu) is exclusively found in families of Portuguese ancestry and is present in approximately 30% of all Portuguese families with hereditary breast and ovarian cancers. We report the generation and characterization of the first iPSC line from a female donor harboring the Portuguese BRCA2 founder mutation. Skin fibroblasts were reprogrammed using a non-integrative Sendai virus. These iPSCs are a valuable tool to study the origin of BRCA2-associated cancer in its earliest phases.

How BRCA1 germline mutations predispose to cancer remains poorly understood. Induced pluripotent stem cells (iPSCs) represent an emerging model to investigate the molecular mechanisms underlying malignant transformation in primary cells from individuals who are carriers of deleterious mutations in the BRCA1 gene. Here we report the generation and characterization of iPSC lines from a female donor harboring a germline c.3612delA mutation in the BRCA1 gene and her daughter who does not carry the mutation. Skin fibroblasts were reprogrammed using non-integrative Sendai virus and characterized for their pluripotent properties. These iPSCs are a valuable cellular model for personalized pre-clinical research in the context of BRCA1 mutant hereditary cancers.

© 2021 Wiley Periodicals LLC, Human-induced pluripotent stem cells (iPSCs) have great potential for disease modeling. However, generating iPSC-derived models to study brain diseases remains a challenge. In particular, the ability to recapitulate cerebellar development in vitro is still limited. We presented a reproducible and scalable production of cerebellar organoids by using the novel single-use Vertical-Wheel bioreactors, in which functional cerebellar neurons were obtained. Here, we evaluate the global gene expression profiles by RNA sequencing (RNA-seq) across cerebellar differentiation, demonstrating a faster cerebellar commitment in this novel dynamic differentiation protocol. Furthermore, transcriptomic profiles suggest a significant enrichment of extracellular matrix (ECM) in dynamic-derived cerebellar organoids, which can better mimic the neural microenvironment and support a consistent neuronal network. Thus, an efficient generation of organoids with cerebellar identity was achieved for the first time in a continuous process using a dynamic system without the need of organoids encapsulation in ECM-based hydrogels, allowing the possibility of large-scale production and application in high-throughput processes. The presence of factors that favors angiogenesis onset was also detected in dynamic conditions, which can enhance functional maturation of cerebellar organoids. We anticipate that large-scale production of cerebellar organoids may help developing models for drug screening, toxicological tests, and studying pathological pathways involved in cerebellar degeneration., This study was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal (UIDB/04565/2020 through Programa Operacional Regional de Lisboa 2020, Project no. 007317, PD/BD/105773/2014 to Teresa P. Silva and SFRH/BD/147906/2019 to Rui Sousa-Luís), projects co-funded by FEDER (POR Lisboa 2020—Programa Operacional Regional de Lisboa PORTUGAL 2020) and FCT through Grant PAC-PRECISE LISBOA-01-0145-FEDER-016394 and CEREBEX Generation of Cerebellar Organoids for Ataxia Research Grant LISBOA-01-0145-FEDER-029298.

SUMMARYMammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly due to technical limitations in purifying authentic nascent transcripts. We present a new approach to purify and profile nascent RNA, called Polymerase Intact Nascent Transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5’ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano) and profiles whole transcription units (POINT-seq). In particular we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5’P-RNA at polyadenylation sites. Furthermore POINT-nano reveals that splicing occurs either immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.HIGHLIGHTSPOINT methodology dissects intact nascent RNA processingSpecificity of Xrn2 exonuclease in co-transcriptional RNA degradationSplicing suppresses Xrn2-dependent premature terminationDifferent kinetic classes of co-transcriptional splicing in human genes

Widespread co-transcriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure co-transcriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nucleotides of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.

Smaug is a conserved translational repressor that recognizes specific RNA motifs in a large number of mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs have been shown to form membraneless organelles (MLOs) in several organisms and cell types. Using single-molecule FISH we show here that SDHB and UQCRC1 mRNAs associate with Smaug1 MLOs in the human cell line U2OS. Simultaneous loss of function of Smaug1 and Smaug2 affects both mitochondrial respiration and mitochondrial network morphology. Deletion of specific Smaug1 protein regions resulted in impaired MLO formation that correlates with mitochondrial defects. In addition, rotenone but not the respiratory chain uncoupling agent CCCP rapidly induces Smaug1 MLO dissolution. Finally, metformin elicits a similar effect on Smaug1 MLOs and provokes the release of bounded mRNAs. We propose that mitochondrial activity affects Smaug1 MLO dynamics, thus allowing for regulation of nuclear mRNAs that encode key mitochondrial proteins. Fil: Fernández Alvarez, Ana Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Thomas, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Pascual, Malena Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Habif, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Pimentel, Jerónimo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Corbat, Agustín Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Pessoa, Joao. Universidad de Lisboa; Portugal Fil: la Spina, Pablo Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Boscaglia, Lara. Fundación Instituto Leloir; Argentina Fil: Plessis, Anne. Sorbonne University; Francia. Université Paris Diderot - Paris 7; Francia Fil: Carmo Fonseca, Maria. Universidad de Lisboa; Portugal Fil: Grecco, Hernan Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Casado Pinna, Marta. Instituto de Biomedicina de Valencia; España Fil: Boccaccio, Graciela Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Splice-switching oligonucleotides (SSOs) have been used to modulate gene expression by interfering with pre-mRNA splicing with the intent to treat disease. For Duchenne muscular dystrophy, splicing modulation has been used to induce the skipping of exon 51 of the dystrophin transcript, allowing the production of a truncated but functional protein. Although oligonucleotide-based therapies are promising, the rapid degradation of oligonucleotides (ONs) by intracellular nucleases has been a major obstacle. Locked nucleic acid (LNA) substitution in SSOs protects oligonucleotides from nuclease degradation and enhances the hybridization properties of the oligo. However, the best optimum size of the oligo depends on the LNA substitution rate. Here we show that 16-mer DNA SSOs with 60% LNA substitution and full phosphorothioate (PS) linkage backbone efficiently induce exon 51 skipping in myogenic cells derived from a DMD patient, allowing expression of the dystrophin protein.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/), Mammalian Native Elongating Transcript sequencing (mNET-seq) is a recently developed technique that generates genome-wide profiles of nascent transcripts associated with RNA polymerase II (Pol II) elongation complexes. The ternary transcription complexes formed by Pol II, DNA template and nascent RNA are first isolated, without crosslinking, by immunoprecipitation with antibodies that specifically recognize the different phosphorylation states of the polymerase large subunit C-terminal domain (CTD). The coordinate of the 3' end of the RNA in the complexes is then identified by high-throughput sequencing. The main advantage of mNET-seq is that it provides global, bidirectional maps of Pol II CTD phosphorylation-specific nascent transcripts and coupled RNA processing at single nucleotide resolution. Here we describe the general pipeline to prepare and analyse high-throughput data from mNET-seq experiments., M.C.-F. acknowledges funding from Fundação para a Ciência e Tecnologia (FCT)/ Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado (UID/BIM/50005/2019), and FCT/FEDER/POR Lisboa 2020, Programa Operacional Regional de Lisboa, PORTUGAL 2020 (LISBOA-01-0145-FEDER-016394).. R.G.M. is supported by FCT grants PTDC/BEX-BID/0395/2014, PTDC/BIA-BID/28441/2017, and UID/BIM/04773/2013 CBMR 1334.

How the splicing machinery defines exons or introns as the spliced unit has remained a puzzle for 30 years. Here, we demonstrate that peripheral and central regions of the nucleus harbor genes with two distinct exon-intron GC content architectures that differ in the splicing outcome. Genes with low GC content exons, flanked by long introns with lower GC content, are localized in the periphery, and the exons are defined as the spliced unit. Alternative splicing of these genes results in exon skipping. In contrast, the nuclear center contains genes with a high GC content in the exons and short flanking introns. Most splicing of these genes occurs via intron definition, and aberrant splicing leads to intron retention. We demonstrate that the nuclear periphery and center generate different environments for the regulation of alternative splicing and that two sets of splicing factors form discrete regulatory subnetworks for the two gene architectures. Our study connects 3D genome organization and splicing, thus demonstrating that exon and intron definition modes of splicing occur in different nuclear regions.

Introduction. Mutations in genes encoding the metabolic enzymes isocitrate dehydrogenase (IDH) 1 and 2 are found in 10-20% of patients with acute myeloid leukemia (AML). Recently, IDH inhibitors have shown good clinical response in patient's refractory to standard treatments, providing evidence for a new treatment paradigm. Comprehensive real-world studies are needed to explore genotype-to-phenotype correlations and prognosis of IDH mutated AML, which may influence targeted treatment strategies. Patients. From a retrospective, European, real-word population (ClinicalTrials.gov Identifier: NCT04369287) we studied 477 IDH mutated patients and 954 IDH wild type patients matched for age, sex and type of treatment with a 1:2 ratio. Results. Median age of IDH mutated patients was 67 years; IDH1 mutations were found in 202 patients (89% carried R132 mutation), while IDH2 mutations were found in 275 cases (51% and 28% carried R140 and R172 mutations, respectively). At diagnosis, IDH mutated patients had lower neutrophil and higher platelet count and higher percentage of marrow blasts (P Considering cytogenetic risk according to ELN criteria, the great majority of IDH1 and IDH2 mutated patients had an intermediate cytogenetic risk (84% and 86%, respectively, P We then analysed the most common co-mutational patterns in IDH mutated patients. A total of 53% of IDH1 mutated patients carried NPM1 mutations (without FLT3 mutations), while the majority of IDH2 mutated patients had wild type NPM1 gene (P Median overall survival from diagnosis (OS) was 14 months for IDH1 mutated patients, 23 for IDH2 mutated patients and 19 for IDH wild type patients (P IDH mutated patients receiving hypomethylating agents (n=211) had a lower response rate vs. wild type patients (56% vs. 36% of treatment failure, respectively, P=.04), while no significant different probability of response to intensive chemotherapy was noticed. In patients who received allogeneic transplantation (n=345), IDH1 mutated patients shower higher relapse rate vs. wild type and IDH2 mutated patients (53% vs. 34%, P Conclusion. In a real world context, AML patients with IDH1 and 2 mutations have high marrow blasts percentage, frequently present normal karyotype and show specific co-mutational patterns with respect to NPM1, FLT3 and ASXL1 genes. IDH1 mutations were an independent predictor of unfavorable outcome with high rate of disease recurrence under currently available treatment options, and could be considered as an additional marker to improve personalized prognostic assessment within ELN risk groups. Dissection of prognosis of IDH mutated AML may influence targeted treatment strategies in clinical practice. Disclosures Voso: Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Heibl:Takeda: Honoraria; AOP orphan: Consultancy, Honoraria, Research Funding; BMS/celgene: Consultancy, Honoraria, Research Funding; novartis: Consultancy, Honoraria. Metzeler:Astellas: Honoraria; Daiichi Sankyo: Honoraria; Otsuka Pharma: Consultancy; Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Honoraria, Research Funding. Thiede:AgenDix GmbH: Other: Co-owner and CEO. Fracchiolla:ABBVIE: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses, Speakers Bureau. Todisco:Jannsen, Abbvie, Jazz: Membership on an entity's Board of Directors or advisory committees. Passamonti:Novartis: Speakers Bureau; BMS: Speakers Bureau; Roche: Other: Support of parent study and funding of editorial support.

Hypertrophic cardiomyopathy (HCM), the most common inherited heart disease, is predominantly caused by mutations in genes that encode sarcomere-associated proteins. Effective gene-based diagnosis is critical for the accurate clinical management of patients and their family members. However, the introduction of high-throughput DNA sequencing approaches for clinical diagnostics has vastly expanded the number of variants of uncertain significance, leading to many inconclusive results that limit the clinical utility of genetic testing. More recently, developments in RNA analysis have been improving diagnostic outcomes by identifying new variants that interfere with splicing. This review summarizes recent discoveries of RNA mis-splicing in HCM and provides an overview of research that aims to apply the concept of RNA therapeutics to HCM.

The cerebellum plays a critical role in all vertebrates, and many neurological disorders are associated with cerebellum dysfunction. A major limitation in cerebellar research has been the lack of adequate disease models. As an alternative to animal models, cerebellar neurons differentiated from pluripotent stem cells have been used. However, previous studies only produced limited amounts of Purkinje cells. Moreover, in vitro generation of Purkinje cells required co-culture systems, which may introduce unknown components to the system. Here we describe a novel differentiation strategy that uses defined medium to generate Purkinje cells, granule cells, interneurons, and deep cerebellar nuclei projection neurons, that self-formed and differentiated into electrically active cells. Using a defined basal medium optimized for neuronal cell culture, we successfully promoted the differentiation of cerebellar precursors without the need for co-culturing. We anticipate that our findings may help developing better models for the study of cerebellar dysfunctions, while providing an advance toward the development of autologous replacement strategies for treating cerebellar degenerative diseases.

Splice-switching oligonucleotides (SSOs) have been used to modulate gene expression by interfering with pre-mRNA splicing with the intent to treat disease. For Duchenne muscular dystrophy, splicing modulation has been used to induce the skipping of exon 51 of the dystrophin transcript, allowing the production of a truncated but functional protein. Although oligonucleotide-based therapies are promising, the rapid degradation of oligonucleotides (ONs) by intracellular nucleases has been a major obstacle. Locked nucleic acid (LNA) substitution in SSOs protects oligonucleotides from nuclease degradation and enhances the hybridization properties of the oligo. However, the best optimum size of the oligo depends on the LNA substitution rate. Here we show that 16-mer DNA SSOs with 60% LNA substitution and full phosphorothioate (PS) linkage backbone efficiently induce exon 51 skipping in myogenic cells derived from a DMD patient, allowing expression of the dystrophin protein.

Copyright © 2019, Springer Nature Limited, Discoveries in the past decade have highlighted the potential of mRNA as a therapeutic target for cancer. Specifically, RNA sequencing revealed that, in addition to gene mutations, alterations in mRNA can contribute to the initiation and progression of cancer. Indeed, precursor mRNA processing, which includes the removal of introns by splicing and the formation of 3' ends by cleavage and polyadenylation, is frequently altered in tumours. These alterations result in numerous cancer-specific mRNAs that generate altered levels of normal proteins or proteins with new functions, leading to the activation of oncogenes or the inactivation of tumour-suppressor genes. Abnormally spliced and polyadenylated mRNAs are also associated with resistance to cancer treatment and, unexpectedly, certain cancers are highly sensitive to the pharmacological inhibition of splicing. This Review summarizes recent progress in our understanding of how splicing and polyadenylation are altered in cancer and highlights how this knowledge has been translated for drug discovery, resulting in the production of small molecules and oligonucleotides that modulate the spliceosome and are in clinical trials for the treatment of cancer., The authors acknowledge funding from Fundação para a Ciência e Tecnologia and FEDER/POR Lisboa 2020 — Programa Operacional Regional de Lisboa, PORTUGAL 2020 (LISBOA-01–0145-FEDER-016394; 007391)

Discoveries in the past decade have highlighted the potential of mRNA as a therapeutic target for cancer. Specifically, RNA sequencing revealed that, in addition to gene mutations, alterations in mRNA can contribute to the initiation and progression of cancer. Indeed, precursor mRNA processing, which includes the removal of introns by splicing and the formation of 3' ends by cleavage and polyadenylation, is frequently altered in tumours. These alterations result in numerous cancer-specific mRNAs that generate altered levels of normal proteins or proteins with new functions, leading to the activation of oncogenes or the inactivation of tumour-suppressor genes. Abnormally spliced and polyadenylated mRNAs are also associated with resistance to cancer treatment and, unexpectedly, certain cancers are highly sensitive to the pharmacological inhibition of splicing. This Review summarizes recent progress in our understanding of how splicing and polyadenylation are altered in cancer and highlights how this knowledge has been translated for drug discovery, resulting in the production of small molecules and oligonucleotides that modulate the spliceosome and are in clinical trials for the treatment of cancer.