Inhibition of human metapneumovirus replication by small interfering RNA.

BACKGROUND: Human metapneumovirus (hMPV) is a major respiratory viral pathogen in young children, elderly individuals and immunocompromised patients. Despite its major effects related to bronchiolitis, pneumonia and its potential role in recurrent wheezing episodes, there is still no commercial treatment or vaccine available against this paramyxovirus. METHODS: We tested a therapeutic strategy for hMPV that was based on RNA interference. RESULTS: An hMPV genome-wide search for small interfering RNAs (siRNAs) by computational analysis revealed 200 potentially effective 21-mer siRNAs. Initial screening with a luciferase assay identified 57 siRNAs of interest. Further evaluation of their inhibitory potential against the four hMPV subgroups by quantitative real-time reverse transcriptase PCR and plaque immunoassay identified two highly potent siRNAs with 50% inhibitory concentration (IC50) values in the subnanomolar range. siRNA45 targets the nucleoprotein messenger RNA (mRNA) and had IC50 values <0.078 nM against representative strains from the four hMPV subgroups, whereas siRNA60, which targets the phosphoprotein mRNA, had IC50 values between 0.090-<0.078 nM against the same panel of hMPV strains. Longer25/27-mer siRNAs known as Dicer substrates designed from the top two siRNA candidates were also evaluated and were at least as effective as their corresponding 21-mer siRNAs. Interestingly, the presence of one or two nucleotide mismatches in the target mRNA sequence of some hMPV subgroups did not always affect hMPV inhibition in vitro. CONCLUSIONS: We successfully identified two highly efficient siRNAs against hMPV targeting essential components of the hMPV replication complex.

Therapeutic utility of a novel tight junction modulating peptide for enhancing intranasal drug delivery.

Previously, a novel tight junction modulating (TJM) peptide was described affording a transient, reversible lowering of transepithelial electrical resistance (TER) in an in vitro model of nasal epithelial tissue. In the current report, this peptide has been further evaluated for utility as an excipient in transepithelial drug formulations. Chemical stability was optimal at neutral to acidic pH when stored at or below room temperature, conditions relevant to therapeutic formulations. The TJM peptide was tested in the in vitro tissue model for potential to enhance permeation of a low-molecular-weight (LMW) drug, namely the acetylcholinesterase inhibitor galantamine, as well as three peptides, salmon calcitonin, parathyroid hormone 1-34 (PTH(1-34)), and peptide YY 3-36 (PYY(3-36)). In all cases, the TJM peptide afforded a dramatic improvement in drug permeation across epithelial tissue. In addition, a formulation containing PYY(3-36) and TJM peptide was dosed intranasally in rabbits, resulting in a dramatic increase in bioavailability. The TJM peptide was as or more effective in enhancing PYY(3-36) permeation in vivo at a 1000-fold lower molar concentration compared to using LMW enhancers. Based on these in vitro and in vivo data, the novel TJM peptide represents a promising advancement in intranasal formulation development.

A functional erythropoietin receptor is necessary for the action of thrombopoietin on erythroid cells lacking c-mpl.

OBJECTIVE: We hypothesized that thrombopoietin (TPO) exerts its mitogenic effects on erythroid cells, at least in part, via an interaction of TPO with the cells' erythropoietin receptor (EPO-R). METHODS: We used BaF3 cells stably transfected with EPO-R to demonstrate that TPO alone is sufficient to support the long-term growth and proliferation of BaF3/EPO-R cells and to develop a TPO-dependent variant, BaF3/EPO-R(T), which is highly sensitive to and dependent on TPO for its proliferation. Northern analysis and RT-PCR were used to verify that both BaF3/EPO-R and BaF3/EPO-R(T) cells express EPO-R but lack c-mpl, the TPO receptor. To confirm that TPO responsiveness of BaF3/EPO-R(T) is due to TPO's interaction with EPO-R, EPO-R was downregulated by antisense mRNA. RESULTS: Downregulation of EPO-R in BaF3/EPO-R(T) cells abolishes responsiveness to both EPO and TPO. Viability of EPO-treated transfectants decreased from 95% to 36%, while that of TPO-treated transfectants decreased from 95% to 9% by 48 hours. Nontransfected BaF3/EPO-R(T), and BaF3/EPO-R(T) transfected with vector alone, remained viable and grew in either EPO or TPO. CONCLUSION: Our results suggest a functional EPO-R may be necessary and sufficient for TPO to exert its mitogenic effects on erythroid cells.