Petit ARN interférent

Les petits ARN interférents (pARNi, ou siRNA pour small interfering RNA) sont de petits ARN pouvant se lier spécifiquement à une séquence d'ARN messagers et ainsi empêcher l'expression de gènes en clivant cet ARN. Les petits ARN interférents sont des ARN interférents au même titre que les miARN Fonctionnement Les petits ARN interférents sont de petits ARN double brin de 21 à 24 nucléotides. Les petits ARN interférents à l'état de double brin sont reconnus dans le cytoplasme de la cellule par un complexe protéique nommé complexe RISC pour RNA-induced silencing complex. Celui-ci s'active en libérant le brin complémentaire de l'ARN ou brin sens. Ce complexe activé va reconnaître son transcrit cible, un ARN messager, par complémentarité des bases nucléiques. Ce système de reconnaissance assure la haute spécificité de ce mécanisme. Une fois la cible liée, la protéine Argonaute, faisant partie du complexe RISC, va couper le transcrit au niveau du site de reconnaissance. Argo es

Identification of Factors of Importance for Spray Drying of Small Interfering RNA-Loaded Lipidoid-Polymer Hybrid Nanoparticles for Inhalation

Background With the recent approval of the first small interfering RNA (siRNA) therapeutic formulated as nanoparticles, there is increased incentive for establishing the factors of importance for the design of stable solid dosage forms of such complex nanomedicines. Methods The aims of this study were: (i) to identify factors of importance for the design of spray-dried siRNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles (LPNs), and (ii) to evaluate their influence on the resulting powders by using a quality-by-design approach. Critical formulation and process parameters were linked to critical quality attributes (CQAs) using design of experiments, and an optimal operating space (OOS) was identified. Results A series of CQAs were identified based on the quality target product profile. The loading (ratio of LPNs to the total solid content) and the feedstock concentration were determined as critical parameters, which were optimized systematically. Mannitol was chosen as stabilizing excipient due to the low water content of the resulting powders. The loading negatively affected the colloidal stability of the LPNs, whereas feedstock concentration correlated positively with the powder particle size. The optimal mannitol-based solid formulation, defined from the OOS, displayed a loading of 5% (w/w), mass median aerodynamic diameter of 3.3 +/- 0.2 mu m, yield of 60.6 +/- 6.6%, and a size ratio of 1.15 +/- 0.03. Dispersed micro-embedded LPNs had preserved physicochemical characteristics as well as in vitro siRNA release profile and gene silencing, as compared to non-spray-dried LPNs. Conclusion The optimal solid dosage forms represent robust formulations suitable for higher scale-up manufacturing.

2019

Identification and functional characterization of protein kinases that modulate Notch signaling under physiological conditions and in cancer

Chhavi Jain

The Notch signaling pathway is a key regulator of cell fate decisions in embryonic development and in adult tissue homeostasis. Mounting evidence suggests that Notch signaling is frequently deregulated in human neoplasms, where depending upon the cellular context it can function both as an oncogene or a tumor suppressor. A plethora of studies shed light on how Notch crosstalks with signaling pathways downstream to manifest either its oncogenic or tumor suppressive activity. However, regulatory networks upstream of the Notch signaling pathway are still poorly understood. Since protein kinases are well-known regulators of a majority of cell signaling pathways, the aim of the current study was to identify novel positive and negative regulatory kinases that modulate Notch signaling. Using a HeLa cell based co-culture assay to read Notch-dependent luciferase activity, a small interference RNA (siRNA) library against the human kinase genome was previously tested in a high-throughput manner. Validation of top candidate kinases from the screening using both siRNA as well as pharmacological inhibitors led to further elimination of false positives and identification of Protein Kinase B (AKT2) and Calmodulin Kinase II (CaMKII) as key positive while Janus kinases (JAKs) as key negative regulators of the Notch signaling pathway. In the first part of the study, we analyzed the positive regulation of AKT2 and CaMKII kinases on Notch signaling in the T-cell acute lymphoblastic leukemia (T-ALL) cells where Notch is oncogenic. It was shown that pharmacological inhibition of either AKT2 or CaMKII kinase in T-ALL cell lines in vitro abrogated the expression of active Notch1 intracellular domain (N1-ICD) as well as of Notch target genes. Moreover, inhibition of these kinases blocked proliferation of T-ALL cells. In the second part of the study, we analyzed the negative regulation of JAK kinases on Notch signaling using a distinct physiological context where Notch is tumor suppressive. It was shown that pharmacological inhibition of JAK kinases led to an induction of Notch receptor transcription and of Notch target genes in the human primary epidermal keratinocytes (HPEK) as well as in the skin squamous cell carcinoma (SCC) cell lines. In addition, the pharmacological inhibition of JAK kinases induced a block in proliferation, cell cycle arrest and differentiation in HPEKs and skin SCCs by increased expression of early differentiation markers. Suppression of Notch signaling in primary keratinocytes counteracted the differentiation-inducing effect of JAK inhibition. Since JAK inhibition affected Notch transcription, global gene expression profiling using RNA sequencing was done to identify transcription factors involved in an indirect regulation of JAK kinases on the Notch cascade. EGR1 and EGR2 belonging to the early growth response family of transcription factors were significantly modulated downstream of JAK inhibition. In vivo, topical inhibition of JAK kinases provided marked resistance against chemically induced cutaneous carcinogenesis.Taken together, our data reveals a key role of AKT2 and CaMKII kinases in positive regulation while of JAK kinases in the negative regulation of Notch signaling, of potential relevance for combinatory approaches for cancer therapy. Pharmacological inhibitors against these kinases could be tested for their ability to modulate Notch signaling and may prove highly beneficial in the therapy of Notch-driven cancers.

EPFL2016

Identification and functional characterization of protein kinases that modulate Notch signaling under physiological conditions and in cancer

Chhavi Jain

EPFL2016

Identification of Factors of Importance for Spray Drying of Small Interfering RNA-Loaded Lipidoid-Polymer Hybrid Nanoparticles for Inhalation

Intracellular delivery of a model drug using monolayer-coated gold nanoparticle carriers and a degredable peptide linker

Identification and functional characterization of protein kinases that modulate Notch signaling under physiological conditions and in cancer

Identification and functional characterization of protein kinases that modulate Notch signaling under physiological conditions and in cancer

Identification of Factors of Importance for Spray Drying of Small Interfering RNA-Loaded Lipidoid-Polymer Hybrid Nanoparticles for Inhalation

Intracellular delivery of a model drug using monolayer-coated gold nanoparticle carriers and a degredable peptide linker