Context-specific synthetic T cell promoters from assembled transcriptional elements.

Genetic engineering of human lymphocytes for therapeutic applications is constrained by a lack of transgene transcriptional control, resulting in a compromised therapeutic index. Incomplete understanding of transcriptional logic limits the rational design of contextually responsive genetic modules1. Here, we juxtaposed rationally curated transcriptional response element (TRE) oligonucleotides by random concatemerization to generate a library from which we selected context-specific inducible synthetic promoters (iSynPros). Through functional selection, we screened an iSynPro library for "IF-THEN" logic-gated transcriptional responses in human CD8+ T cells expressing a 4-1BB second generation chimeric antigen receptor (CAR). iSynPros exhibiting stringent off-states in quiescent T cells and CAR activation-dependent transcriptional responsiveness were cloned and subjected to TRE composition and pattern analysis, as well as performance in regulating candidate antitumor potency enhancement modules. These data reveal synthetic TRE grammar can mediate logic-gated transgene transcription in human T cells that, when applied to CAR T cell engineering, enhance potency and improve therapeutic indices.

microRNA-1 regulates sarcomere formation and suppresses smooth muscle gene expression in the mammalian heart.

microRNA-1 (miR-1) is an evolutionarily conserved, striated muscle-enriched miRNA. Most mammalian genomes contain two copies of miR-1, and in mice, deletion of a single locus, miR-1-2, causes incompletely penetrant lethality and subtle cardiac defects. Here, we report that deletion of miR-1-1 resulted in a phenotype similar to that of the miR-1-2 mutant. Compound miR-1 knockout mice died uniformly before weaning due to severe cardiac dysfunction. miR-1-null cardiomyocytes had abnormal sarcomere organization and decreased phosphorylation of the regulatory myosin light chain-2 (MLC2), a critical cytoskeletal regulator. The smooth muscle-restricted inhibitor of MLC2 phosphorylation, Telokin, was ectopically expressed in the myocardium, along with other smooth muscle genes. miR-1 repressed Telokin expression through direct targeting and by repressing its transcriptional regulator, Myocardin. Our results reveal that miR-1 is required for postnatal cardiac function and reinforces the striated muscle phenotype by regulating both transcriptional and effector nodes of the smooth muscle gene expression network. DOI: http://dx.doi.org/10.7554/eLife.01323.001.