Lipidated conjugates for CNS delivery

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FGF-4 signaling is involved in mir-206 expression in developing somites of chicken embryos

The microRNAs (miRNAs) are recently discovered short, noncoding RNAs, that regulate gene expression in metazoans. We have cloned short RNAs from chicken embryos and identified five new chicken miRNA genes. Genome analysis identified 17 new chicken miRNA genes based on sequence homology to previously characterized mouse miRNAs. Developmental Northern blots of chick embryos showed increased accumulation of most miRNAs analyzed from 1.5 days to 5 days except, the stem cell-specific mir-302, which was expressed at high levels at early stages and then declined. In situ analysis of mature miRNAs revealed the restricted expression of mir-124 in the central nervous system and of mir-206 in developing somites, in particular the developing myotome. In addition, we investigated how miR-206 expression is controlled during somite development using bead implants. These experiments demonstrate that fibroblast growth factor (FGF) -mediated signaling negatively regulates the initiation of mir-206 gene expression. This may be mediated through the effects of FGF on somite differentiation. These data provide the first demonstration that developmental signaling pathways affect miRNA expression. Thus far, miRNAs have not been studied extensively in chicken embryos, and our results show that this system can complement other model organisms to investigate the regulation of many other miRNAs

A era dos medicamentos de ARN interferência: o panorama clínico dos fármacos para silenciamento génico

ABSTRACT - Therapeutic oligonucleotides, such as small interfering RNAs (siRNAs), provide a simple and effective tool to modulate the expression of any gene. siRNAs harness the RNA interference (RNAi) pathway to degrade disease-associated messenger RNAs (mRNAs). The inherent sequence specificity and potency of siRNAs makes them ideal drug candidates that are expected to transform drug development and our approach to human health. However, the first wave of clinical trials was not immediately successful and temporarily dampened the excitement over this newly discovered technology. Most studies did not meet the desired efficacy and failed to achieve clinically-relevant endpoints. Poor chemical design, lack of enzymatic stability and inadequate delivery strategies were found to be the main issues stifling success. Recent advancements in RNA chemistry, biology, and mechanistic understanding of factors that define oligonucleotide pharmacokinetic/pharmacodynamic behavior have resulted in a fundamental shift in the clinical landscape of this novel class of therapeutic modalities. As a result, there has been a dramatic increase in both the numbers of clinical trials and, more importantly, the level of observed clinical efficacy. In 2018, we witnessed a major landmark for the field with the first RNAi-based therapeutics, Patisiran (OnpattroTM), being approved by the Food and Drug Administration and the European Medicines Agency. Several other lead compounds have achieved unprecedented levels of activity following a single treatment dose, and hold great promise as new therapeutic strategies for liver diseases. This review gives an overview of the clinical landscape of synthetic RNAi drugs, contextualizing how advances in RNAi chemistry and formulation strategies have helped define the clinical utility of this promising class of drugs.RESUMO - Oligonucleotídeos sintéticos, como os small interfering RNAs (siRNAs), providenciam uma forma simples e eficiente de modular a expressão de qualquer gene. siRNAs utilizam um mecanismo endógeno, chamado ARN interferência, para degradar ARN mensageiros que estejam associados a condições patológicas. A capacidade de silenciar genes com elevada especificidade e potência faz dos siRNAs fármacos ideais com um elevado potencial para transformar o ramo da medicina e a forma como se faz desenvolvimento farmacêutico. Contudo, os primeiros ensaios clínicos com esta tecnologia não tiveram sucesso imediato, o que reduziu temporariamente o entusiasmo da comunidade científica. A maioria destes estudos iniciais não atingiram a eficácia clínica desejada. A introdução prematura de fármacos que não se encontravam devidamente estabilizados e a utilização de estratégias de administração inadequadas foram as principais causas dos primeiros fracassos. Avanços recentes na síntese química de oligonucleotídeos, como a melhor compreensão dos processos biológicos que definem a farmacocinética/farmacodinâmica destes fármacos, resultou numa mudança drástica no panorama clínico desta nova modalidade terapêutica. O número de ensaios clínicos tem aumentado significativamente ao longo dos últimos anos, em paralelo com o aumento da eficácia terapêutica destes medicamentos. Em 2018 foi testemunhado um marco importante para o ramo de desenvolvimento destes fármacos, com a aprovação do primeiro produto baseado em liposomas, Patisiran (OnpattroTM), pela Food and Drug Administration e pela European Medicines Agency. Aproximadamente um ano mais tarde foi aprovado o primeiro fármaco que utiliza a estratégia de conjugados que possibilita a internalização específica em hepatócitos, Givosiran (GIVLAARITM). A recente aprovação destes dois fármacos trouxe uma esperança renovada ao ramo de ARN interferência, alimentando o interesse nesta estratégia como poderosa ferramenta terapêutica para doenças do foro genético. Este artigo de revisão pretende providenciar uma perspetiva geral sobre o panorama clínico dos fármacos para silenciamento génico, contextualizando o papel dos avanços tecnológicos que permitiram a definição desta modalidade como uma nova classe farmacêutica.info:eu-repo/semantics/publishedVersio

Ribozyme-based insulator parts buffer synthetic circuits from genetic context

Synthetic genetic programs are built from circuits that integrate sensors and implement temporal control of gene expression. Transcriptional circuits are layered by using promoters to carry the signal between circuits. In other words, the output promoter of one circuit serves as the input promoter to the next. Thus, connecting circuits requires physically connecting a promoter to the next circuit. We show that the sequence at the junction between the input promoter and circuit can affect the input-output response (transfer function) of the circuit. A library of putative sequences that might reduce (or buffer) such context effects, which we refer to as 'insulator parts', is screened in Escherichia coli. We find that ribozymes that cleave the 5′ untranslated region (5′-UTR) of the mRNA are effective insulators. They generate quantitatively identical transfer functions, irrespective of the identity of the input promoter. When these insulators are used to join synthetic gene circuits, the behavior of layered circuits can be predicted using a mathematical model. The inclusion of insulators will be critical in reliably permuting circuits to build different programs.Life Technologies, Inc.United States. Defense Advanced Research Projects Agency (DARPA CLIO N66001-12-C-4018)United States. Office of Naval Research (N00014-10-1-0245)National Science Foundation (U.S.) (CCF-0943385)National Institutes of Health (U.S.) (AI067699)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SynBERC, SA5284-11210

Computational Design of Artificial RNA Molecules For Gene Regulation

This volume provides an overview of RNA bioinformatics methodologies, including basic strategies to predict secondary and tertiary structures, and novel algorithms based on massive RNA sequencing. Interest in RNA bioinformatics has rapidly increased thanks to the recent high-throughput sequencing technologies allowing scientists to investigate complete transcriptomes at single nucleotide resolution. Adopting advanced computational technics, scientists are now able to conduct more in-depth studies and present them to you in this book. Written in the highly successful Methods of Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and equipment, step-by-step, readily reproducible bioinformatics protocols, and key tips to avoid known pitfalls.Authoritative and practical, RNA Bioinformatics seeks to aid scientists in the further study of bioinformatics and computational biology of RNA