Development and Optimization of Logic Gated Small Interfering RNAs for Operation Inside Mammalian Cells

In 1900, Paul Ehrlich put forth the concept of a pharmacologic “magic bullet”, a combination of a disease selective chemical agent conjugated to a non-selective toxin that could target drug activity to disease causing cells without harming healthy tissues in the body. For the past two decades, various researchers in DNA and RNA nanotechnology have proposed ideas for riboswitch regulated magic bullets in which the activities of oligonucleotide drugs are switched ON or OFF according to the presence or absence of specific biomarkers in tissues or cells. Although elegant in concept, practical implementation of these “logical therapeutics” for mammalian cells has proven elusive. We have now developed a conditionally activated small interfering RNA (Cond-siRNA) in which an RNA sensor regulates the RNAi activity of a conjugated siRNA post-transfection into mammalian cells. The sensor switches ON RNAi activity when it base-pairs to an RNA transcript from a designated “trigger gene”. In cells without trigger gene expression, the sensor keeps RNAi activity OFF. The identities of the trigger and target genes are encoded by two entirely independent, easily programmable RNA sequences, giving the Cond-siRNAs the ability to target a specific population of cells for the silencing of an arbitrary gene. We will present experimental data for Cond-siRNAs with different trigger and target gene combinations in different mammalian cell lines, explain the design principles and optimizations that enable easy programmability and correct functioning in mammalian cells, and demonstrate an early example of therapeutic application development in the treatment of cardiac hypertrophy. As small, chemically modified, unimolecular RNA complexes, Cond-siRNAs are compatible with existing methods of siRNA delivery and practical for development into clinically viable RNAi drugs. By making possible post-delivery targeting of RNAi activity to specific populations of disease driving cells, they could enable paradigm shifting development of RNAi drugs that can safely target pleiotropic genes whose dysregulated activities drive the progression of many chronic diseases lacking effective treatments today

BRAF V600E-induced senescence drives Langerhans cell histiocytosis pathophysiology

Langerhans cell histiocytosis (LCH) is a potentially fatal condition characterized by granulomatous lesions with characteristic clonal mononuclear phagocytes (MNPs) harboring activating somatic mutations in mitogen-activated protein kinase (MAPK) pathway genes, most notably BRAFV600E. We recently discovered that the BRAFV600E mutation can also affect multipotent hematopoietic progenitor cells (HPCs) in multisystem LCH disease. How the BRAFV600E mutation in HPCs leads to LCH is not known. Here we show that enforced expression of the BRAFV600E mutation in early mouse and human multipotent HPCs induced a senescence program that led to HPC growth arrest, apoptosis resistance and a senescence-associated secretory phenotype (SASP). SASP, in turn, promoted HPC skewing toward the MNP lineage, leading to the accumulation of senescent MNPs in tissue and the formation of LCH lesions. Accordingly, elimination of senescent cells using INK-ATTAC transgenic mice, as well as pharmacologic blockade of SASP, improved LCH disease in mice. These results identify senescent cells as a new target for the treatment of LCH