Current viral vectors for gene therapy are associated with serious safety concerns, including leukemogenesis, and nonviral vectors are limited by low gene transfer efficiency. Here we investigate the therapeutic utility of chemically modified mRNA as an alternative to DNA-based gene therapy. A combination of nucleotide modifications abrogates mRNA interaction with Toll-like receptor (TLR)3, TLR7, TLR8 and retinoid-inducible gene I (RIG-I), resulting in low immunogenicity and higher stability in mice. A single intramuscular injection of modified murine erythropoietin mRNA raises the average hematocrit in mice from 51.5% to 64.2% after 28 days. In a mouse model of a lethal congenital lung disease caused by a lack of surfactant protein B (SP-B), twice weekly local application of an aerosol of modified SP-B mRNA to the lung restored 71% of the wild-type SP-B expression, and treated mice survived until the predetermined end of the study after 28 days.

The inhalation of medical aerosols is widely used for the treatment of lung disorders such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, respiratory infection and, more recently, lung cancer. Targeted aerosol delivery to the affected lung tissue may improve therapeutic efficiency and minimize unwanted side effects. Despite enormous progress in optimizing aerosol delivery to the lung, targeted aerosol delivery to specific lung regions other than the airways or the lung periphery has not been adequately achieved to date. Here, we show theoretically by computer-aided simulation, and for the first time experimentally in mice, that targeted aerosol delivery to the lung can be achieved with aerosol droplets comprising superparamagnetic iron oxide nanoparticles--so-called nanomagnetosols--in combination with a target-directed magnetic gradient field. We suggest that nanomagnetosols may be useful for treating localized lung disease, by targeting foci of bacterial infection or tumour nodules.

The Na-K-Cl cotransporter NKCC1 is activated by phosphorylation of a regulatory domain in its N terminus. In the accompanying paper (Darman, R. B., and Forbush, B. (2002) J. Biol. Chem. 277, 37542-37550), we identify three phosphothreonines important in this process. Using a phospho-specific antibody (anti-phospho-NKCC1 antibody R5) raised against a diphosphopeptide containing Thr 212 and Thr 217 of human NKCC, we were readily able to monitor the cotransporter activation state. In 32 P phosphorylation experiments with rectal gland tubules, we show that the R5 antibody signal is proportional to the amount of 32 P incorporated into NKCC1; and in experiments with NKCC1-transfected HEK-293 cells, we demonstrate that R5-detected phosphorylation directly mirrors functional activation. Immunofluorescence analysis of shark rectal gland shows activation-dependent R5 antibody staining along the basolateral membrane. In perfused rat parotid glands, isoproterenol induced staining of both acinar and ductal cells along the basolateral membrane. Isoproterenol also induced basolateral staining of the epithelial cells in rat trachea, whereas basal cells in the subepithelial tissue displayed heavy, non-polarized staining of the cell membrane. In rat colon, agonist stimulation induced staining along the basolateral membrane of crypt cells. These data provide direct evidence of NKCC1 regulation in these tissues, and they further link phosphorylation of NKCC1 with its activation in transfected cells and native tissue. The high conservation of the regulatory threonine residues among NKCC1, NKCC2, and NCC family members, together with the fact that tissues from divergent vertebrate species exhibit similar R5-binding profiles, lends further support to the role of this regulatory locus in vivo. NKCC1, the secretory or housekeeping isoform of the Na-K-Cl cotransporter, is expressed in most cell types, aiding in the regulation of cell volume. In polarized cells of secretory epithelia, NKCC1 is heavily expressed along the basolateral membrane, activated in response to secretagogues, and paramount for the transepithelial secretion of Cl Ϫ and water (2). NKCC1-mediated Cl Ϫ secretion has been well documented in rat parotid gland (3, 4), shark rectal gland (5), rat colon (6), and dog trachea (7). At least in shark rectal gland, the evidence is consistent with an indirect activation of NKCC1 upon agonist stimulation: secretagogues cause a drop in intracellular [Cl Ϫ ] and volume via protein kinase A-mediated phosphorylation of apical chloride channels; in turn, low intracellular [Cl Ϫ ] and low cell volume provide activation stimuli for the currently unidentified NKCC1 kinase(s) (2, 8).Our laboratory (9) and others (10) have linked the phosphorylation of the intracellular N-terminal domain with NKCC1 activation. In the accompanying paper, Darman and Forbush (1) describe the phosphorylation of three residues in this regulatory domain, of which Thr 184 and Thr 189 are necessary for maximal sNKCC1 1 activation. Thr 189 in particular is demonstra...

The specificity of major protein phosphatases is conferred via targeting subunits, each of which binds specifically to the phosphatase and targets it to the vicinity of substrate proteins. In the case of protein phosphatase 1 (PP1), an RVXFXD motif on a targeting subunit binds to a cleft in PP1c, the catalytic subunit. Here we report that a substrate of PP1, the Na-K-Cl cotransporter (NKCC1), bears this motif in its N terminus near sites of regulatory phosphorylation and that direct binding of PP1 to NKCC1 is functionally important in determining the set point for intracellular chloride regulation. NKCC1 mutants in which the motif is destroyed or improved exhibit dramatically shifted activation curves because of a change in the rate of cotransporter dephosphorylation. Furthermore, direct interaction of NKCC1 and PP1c observed by coprecipitation of the two proteins is not seen in a mutant lacking the site. This establishes a new paradigm of phosphatase specificity, one in which a substrate protein containing an RVXFXD motif binds directly to PP1c; we propose that this may be a quite general mechanism.A small number of protein phosphatases is responsible for dephosphorylation of the majority of cellular phosphoproteins (1, 2). The specificity of these otherwise promiscuous enzymes is conferred by targeting subunits, each of which binds specifically to the phosphatase and targets it to the vicinity of substrate proteins. For protein phosphatase 1 (PP1), 1 a cleft in the catalytic subunit (PP1c) binds proteins that contain the consensus motif, RVXFXD, illustrated both in a direct examination of the molecular structure (3) and by the results of peptide panning experiments (4). The present study began with the observation that the N terminus of the Na-K-Cl cotransporter (NKCC1) contains a highly conserved region with the sequence RVNFVD (residues 140 -145 in human NKCC1, 107-112 in shark NKCC1), posing the possibility that the cotransporter is directly targeted as a phosphatase substrate. The Na-K-Cl cotransporter is an integral membrane protein constituting a major regulated pathway for coupled inward movement of Na ϩ , K ϩ , and Cl Ϫ in many cell types. The transporter is a central element in electrolyte movement by secretory epithelia, where it functions in concert with cystic fibrosis transmembrane conductance regulator, potassium channels, and the sodium pump to bring about transcellular chloride transport (5, 6). Cotransport activity is fully controlled by direct phosphorylation of the NKCC1 protein (7-9) in response to decreases in cell volume or intracellular [Cl]. Phosphorylation of NKCC1 is mediated by a kinase whose identity is still unknown, but dephosphorylation appears to occur through the action of protein phosphatase 1 (10). The phosphoregulatory region, including three identified threonine phosphoacceptors, is in the cytoplasmic N terminus of the NKCC1 (7, 11-13). Functional characteristics of cotransporter regulation are highly conserved among vertebrates from shark to human (14), consistent with con...

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