No abstract

Rhinovirus (RV) infections are the major cause of asthma exacerbations, the major cause of morbidity and mortality in asthma. MUC5AC is the major mucin produced by bronchial epithelial cells. Whether RV infection upregulates MUC5AC in vivo is unknown and the molecular mechanisms involved are incompletely understood.We investigated RV induction of MUC5AC in vivo and in vitro to identify targets for development of new therapies for asthma exacerbations.RV infection increased MUC5AC release in normal and asthmatic volunteers experimentally infected with RV-16, and in asthmatic, but not normal, subjects, this was related to virus load. Bronchial epithelial cells were confirmed a source of MUC5AC in vivo. RV induction of MUC5AC in bronchial epithelial cells in vitro occurred via nuclear factor-kB-dependent induction of matrix metalloproteinase-mediated transforming growth factor-a release, thereby activating an epidermal growth factor receptor-dependent cascade culminating, via mitogen-activated protein kinase activation, in specificity protein-1 transactivation of the MUC5AC promoter.RV induction of MUC5AC may be an important mechanism in RV-induced asthma exacerbations in vivo. Revealing the complex serial signalling cascade involved identifies targets for development of pharmacologic intervention to treat mucus hypersecretion in RV-induced illness.

With many safety and technical limitations partly mitigated through chemical modifications, antisense oligonucleotides (ASOs) are gaining recognition as therapeutic entities. The increase in potency realized by ‘third generation chemistries’ may, however, simultaneously increase affinity to unintended targets with partial sequence complementarity. However, putative hybridization-dependent off-target effects (OTEs), a risk historically regarded as low, are not being adequately investigated. Here we show an unexpectedly high OTEs confirmation rate during screening of fully phosphorothioated (PS)-LNA gapmer ASOs designed against the BACH1 transcript. We demonstrate in vitro mRNA and protein knockdown of off-targets with a wide range of mismatch (MM) and gap patterns. Furthermore, with RNase H1 activity residing within the nucleus, hybridization predicted against intronic regions of pre-mRNAs was tested and confirmed. This dramatically increased ASO-binding landscape together with relatively high potency of such interactions translates into a considerable safety concern. We show here that with base pairing-driven target recognition it is possible to predict the putative off-targets and address the liability during lead design and optimization phases. Moreover, in silico analysis performed against both primary as well as spliced transcripts will be invaluable in elucidating the mechanism behind the hepatoxicity observed with some LNA-modified gapmers.

Antisense oligonucleotides (ASOs) modulate cellular target gene expression through direct binding to complementary RNA. Advances in ASO chemistry have led to the development of phosphorothioate (PS) ASOs with constrained-ethyl modifications (cEt). These next-generation cEt-ASOs can enter cells without transfection reagents. Factors involved in intracellular uptake and trafficking of cEt-ASOs leading to successful target knockdown are highly complex and not yet fully understood. AZD4785 is a potent and selective therapeutic KRAS cEt-ASO currently under clinical development for the treatment of cancer. Therefore, we used this to investigate mechanisms of cEt-ASO trafficking across a panel of cancer cells. We found that the extent of ASO-mediated KRAS mRNA knockdown varied significantly between cells and that this did not correlate with bulk levels of intracellular accumulation. We showed that in cells with good productive uptake, distribution of ASO was perinuclear and in those with poor productive uptake distribution was peripheral. Furthermore, ASO rapidly trafficked to the late endosome/lysosome in poor productive uptake cells compared to those with more robust knockdown. An siRNA screen identified several factors mechanistically involved in productive ASO uptake, including the endosomal GTPase Rab5C. This work provides novel insights into the trafficking of cEt-ASOs and mechanisms that may determine their cellular fate.

We have analyzed the 5'-flanking region of one of the genes coding for the human acute-phase protein, serum amyloid A (SAA). We found that SAA mRNA could be increased fivefold in transfected cells by treatment with phorbol 12-myristate 13-acetate (PMA). To analyze this observation further, we placed a 265-base-pair 5' SAA fragment upstream of the reporter chloramphenicol acetyltransferase (CAT) gene and transfected this construct into HeLa cells. PMA treatment of these transient transfectants resulted in increased CAT expression. Nuclear proteins from PMA-treated HeLa cells bound to this DNA fragment, and methylation interference analysis showed that the binding was specific to the sequence GGGACTTTCC (between -82 and -91), a sequence previously described by R. Sen and D. Baltimore (Cell 46:705-716, 1986) Human serum amyloid A (SAA) is a major acute-phase reactant produced mainly by the liver. During periods of inflammation and tissue damage, serum levels of SAA can increase by up to 1,000-fold. SAA is also the precursor peptide of the amyloid A protein subunit of amyloid fibrils in secondary, or reactive, amyloidosis (24). Insoluble fibrils are deposited extracellularly in multiple organs, compromising their normal function. This is a serious complication of chronic or recurrent inflammatory conditions, e.g., juvenile chronic arthritis, in which persistently high serum levels of SAA are found. More than one human SAA gene exist (16,25,41), and in mice three active SAA genes and a pseudogene have been described (29,51). Both human and murine SAA gene expression can be induced by the cytokines interleukin-1 (IL-1) and tumor necrosis factor (39,49 (42). DNA fragments from the 5'-flanking region of the SAA genomic clone, SAAg9 (49), were subcloned into M13mpl8 and -mpl9 and sequenced by using sequence-specific oligonucleotides generated on an Applied Biosystems automated nucleotide synthesizer.Plasmid-constructions. A 265-base-pair (bp) Sau3A DNA fragment, from the promoter region and 33 bp of the first exon of the human SAA gene, was cloned between the BglII-BamHI sites of the vector, pTK.CAT3 (31). This construct contains the entire CAT gene, and the thymidine kinase promoter has been replaced with the SAA 5'-flanking region. As a control, the thymidine kinase promoter was deleted from pTK.CAT3 with BglII-BamHI. The subsequent constructs, OCAT/265 and OCAT, were used to study PMA inducibility conferred on the CAT gene by the SAA promoter region.The 265-bp promoter fragment was also cloned into the BamHI site of the vector pBluescriptSK, generating plasmid 9-2. This vector contains both T3 and T7 promoters flanking the cloned insert, and it was used to generate both codingand noncoding-strand cRNA probes used for RNase mapping.Wild-type (AGGGACTTTCC) and mutant (ACTCACTT TCC) NFKB-binding sequences from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) (32), cloned into plasmid pGEM, were kindly provided by G. Nabel for cotransfection-competition studies.Cell lines and DNA transfections....