Large RNAs and ribonucleoprotein complexes have powerful therapeutic potential, but effective cell-targeted delivery tools are limited. Aptamers that internalize into target cells can deliver siRNAs (<15 kDa, 19–21 nt/strand). We demonstrate a modular nanostructure for cellular delivery of large, functional RNA payloads (50–80 kDa, 175–250 nt) by aptamers that recognize multiple human B cell cancer lines and transferrin receptor-expressing cells. Fluorogenic RNA reporter payloads enable accelerated testing of platform designs and rapid evaluation of assembly and internalization. Modularity is demonstrated by swapping in different targeting and payload aptamers. Both modules internalize into leukemic B cell lines and remained colocalized within endosomes. Fluorescence from internalized RNA persists for ≥2 h, suggesting a sizable window for aptamer payloads to exert influence upon targeted cells. This demonstration of aptamer-mediated, cell-internalizing delivery of large RNAs with retention of functional structure raises the possibility of manipulating endosomes and cells by delivering large aptamers and regulatory RNAs.
Peptide amphiphile micelles (PAMs) are attractive vehicles for the delivery of a variety of therapeutic and prophylactic peptides. However, a key limitation of PAMs is their lack of preferential targeting ability. In this paper, we describe our design of a PAM system that incorporates a DNA oligonucleotide amphiphile (antitail amphiphile-AA) to form A/PAMs. A cell-targeting DNA aptamer with a 3' extension sequence (tail) complementary to the AA is annealed to the surface to form aptamer-displaying PAMs (Aptamer~A/PAMs). Aptamer~A/PAMs are small, anionic, stable nanoparticles capable of delivering a large mass percentage peptide amphiphile (PA) compared to targeting DNA components. Aptamer~A/PAMs are stable for over 4 h in the presence of biological fluids. Additionally, the aptamer retains its cell-targeting properties when annealed to the A/PAM, thus leading to enhanced delivery to a specifically-targeted B-cell leukemia cell line. This exciting modular technology can be readily used with a library of different targeting aptamers and PAs, capable of improving the bioavailability and potency of the peptide cargo.
The scaffold molecule POSH is crucial for the regulation of proliferation and effector function in CD8+ T cells. However, its role in CD4+ T cells is not known. Here we found that, disruption of the POSH scaffold complex established a transcriptional profile that strongly skewed differentiation towards TH2, led to decreased survival and had no affect on cell cycle entry. This is in stark contrast to CD8+ T cells where POSH regulates cell cycle and does not affect survival. Disruption of POSH in CD4+ T cells resulted in the loss of Tak1 dependent activation of JNK1/2 and Tak1 mediated survival. However, in CD8+ T cells, POSH regulates only JNK1. Remarkably, each type of T cell had a unique composition of the POSH scaffold complex and distinct post-translational modifications of POSH. These data indicate that the mechanism that regulates POSH function in CD4+ T cells is different from CD8+ T cells. All together, these data strongly suggest that POSH is essential for the integration of cell-type specific signals that regulate the differentiation, survival and function of T cells.
Interleukin-10 (IL-10) mediates an anti-inflammatory response that is executed through the expression of IL-10-induced genes. Certain IL-10-induced genes, exemplified by TNIP3, are induced by IL-10 only in conjunction with a pro-inflammatory signal. We sought to characterize the mechanism whereby IL-10 and Toll-like receptor signals synergized to induce expression of genes like TNIP3 in macrophages. Stimulation with IL-10 and lipopolysaccharide (LPS) synergistically induced an increase in the transcription rate of TNIP3, while having no effect on its mRNA stability. This transcriptional mechanism proved to be generalizable to 14 other genes that also were synergistically induced by IL-10 and LPS in monocytes/macrophages. Although all of the genes had this synergistic transcriptional regulation in common, they could be divided into three subsets based on their differential requirements for de novo protein synthesis and kinetics of expression: namely, primary response genes, early secondary response genes, and late secondary response genes. This coordinated and temporal pattern of transcriptional regulation in response to IL-10 and LPS was conserved in both human and mouse monocytes/macrophages, and it was associated with differential dependencies on PI3K and JNK signaling pathways. These results underscore the complex nature of the IL-10-induced transcriptional response that occurs specifically in LPS-triggered macrophages.
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