RESUMO
The advent of therapeutic mRNAs significantly increases the possibilities of protein-based biologics beyond those that can be synthesized by recombinant technologies (eg, monoclonal antibodies, extracellular enzymes, and cytokines). In addition to their application in the areas of vaccine development, immune-oncology, and protein replacement therapies, one exciting possibility is to use therapeutic mRNAs to program undesired, diseased cells to synthesize a toxic intracellular protein, causing cells to self-destruct. For this approach to work, however, methods are needed to limit toxic protein expression to the intended cell type. Here, we show that inclusion of microRNA target sites in therapeutic mRNAs encoding apoptotic proteins, Caspase or PUMA, can prevent their expression in healthy hepatocytes while triggering apoptosis in hepatocellular carcinoma cells.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , RNA Mensageiro/genética , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Caspases/genética , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Camundongos , MicroRNAs/uso terapêutico , Cultura Primária de Células , Proteínas Proto-Oncogênicas/genética , Células RAW 264.7 , RNA Mensageiro/uso terapêuticoRESUMO
Recent discoveries that provide a link between inositol phosphate (IP) signaling and fundamental cellular processes evoke many exciting new hypotheses about IP function, and underscore the importance of understanding how IP synthesis is regulated. Central to studies of IP metabolism is the essential development of efficient, fast, and reproducible methods for quantitative analysis of IPs in systems ranging from simple cell cultures to more complex tissues and whole organisms. Additionally, in many cases there is a need to pharmacologically and/or genetically alter IP kinase and phosphatase activities in order to visualize low abundance inositol signaling messengers. Here, we describe updated methods for rapid analysis of IP metabolism in normal and genetically manipulated Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Mus musculus, and Homo sapiens.