Chemically modified mRNA is capable of inducing therapeutic levels of protein expression while circumventing the threat of genomic integration often associated with viral vectors. We utilized this novel therapeutic tool to express the regulatory T cell transcription factor, FOXP3, in a time-and site-specific fashion in murine lung, in order to prevent allergic asthma in vivo. We show that modified Foxp3 mRNA rebalanced pulmonary T helper cell responses and protected from allergen-induced tissue inflammation, airway hyperresponsiveness, and goblet cell metaplasia in 2 asthma models. This protection was conferred following delivery of modified mRNA either before or after the onset of allergen challenge, demonstrating its potential as both a preventive and a therapeutic agent. Mechanistically, FOXP3 induction controlled Th2 and

Inactivation of the ubiquitin ligase E6 associated protein (E6AP) encoded by the UBE3A gene has been associated with development of the Angelman syndrome. Recently, it was reported that in mice, loss of E6AP expression results in increased levels of the synaptic protein Arc and a concomitant impaired synaptic function, providing an explanation for some phenotypic features of Angelman syndrome patients. Accordingly, E6AP has been shown to negatively regulate activity-regulated cytoskeleton-associated protein (Arc) and it has been suggested that E6AP targets Arc for ubiquitination and degradation. In our study, we provide evidence that Arc is not a direct substrate for E6AP and binds only weakly to E6AP, if at all. Furthermore, we show that down-regulation of E6AP expression stimulates estradiol-induced transcription of the Arc gene. Thus, we propose that Arc protein levels are controlled by E6AP at the transcriptional rather than at the posttranslational level.

In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency (2015) Nature Biotechnology, 33 (6), pp. 584-586.In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiencyTo the Editor:Nuclease-mediated genome editing holds great potential to knock out or repair diseasecausing genes. An ideal nuclease delivery vehicle is short-lived, does not integrate into the genome, and can enter target cells efficiently. These requirements have not yet been achieved simultaneously by any nuclease delivery vector. We and others have used modified mRNA, which is non-integrating and provides a transient pulse of protein expression, as an alternative to traditional viral vectors [1][2][3][4][5] . This approach allowed us to deliver therapeutic proteins in mouse models of Surfactant Protein B (SP-B) deficiency 3 and experimental asthma 4 . Here we apply it to deliver site-specific nucleases, demonstrating the value of nuclease-encoding chemically modified (nec) mRNA as a tool for in vivo genome editing. We chose a well-established transgenic mouse model of SP-B deficiency 6 in which SP-B cDNA is under the control of a tetracycline-inducible promoter 7 . Administration of doxycycline drives SP-B expression levels similar to those in wild-type mice (Supplementary Fig. 1), whereas cessation of doxycycline leads to phenotypic changes similar to those of the human disease, including thickened alveolar walls, heavy cellular infiltration, increased macrophages and neutrophils, interstitial edema, augmented cytokines in the lavage, a decline in lung function, and fatal respiratory distress leading to death within days 8,9 . We inserted a constitutive CAG promoter immediately upstream of the SP-B cDNA to allow doxycycline-independent expression and prolonged life in treated mice.First, we customized a panel of ZFNs and TALENs targeting the transgenic SP-B cassette ( Fig. 1a and Supplementary Fig. 2). We chose TALEN #1 (T1) and ZFN #3 (Z3) owing to their high activity and proximity to the desired site of promoter integration (Figs. 1a,b; amino acid sequences in Supplementary Fig. 4) and compared delivery by plasmid 1 DNA and mRNA. mRNA delivery resulted in higher levels of double-strand break (DSB)-induction ( Fig. 1c and Supplementary Fig. 3; P < 0.05) and homology-directed repair (HDR) ( Fig. 1d, P < 0.05). As Z3 mRNA was more efficient than T1 mRNA in both cases, Z3 was chosen for further experimentation. Comparison with a Z3-encoding AAV serotype 6 vector (AAV6) ("Z3 AAV") shows the relatively transient expression of Z3 mRNA (Fig. 1e), limiting the time during which off-target cleavage activity could occur.To optimize Z3 expression in the mouse lung, we administered a panel of 3xFLAG-tagged Z3 mRNAs with various modification schemes 2,5,10 , with or without complexation to biocompatible, biodegradable nanoparticles (NPs) made of chitosan-coated poly (lactic-coglycolic) acid (chit-PLGA) 11,12 . Following intratracheal (i.t.) delivery, NP-complexation significantly increased mRNA expression levels ( Supplem...

Infection rates of Ixodes ticks with Borrelia miyamotoi in Europe and the United States vary greatly based upon location.

Ticks harbor numerous pathogens of significance to human and animal health. A better understanding of the pathogens carried by ticks in a given geographic area can alert health care providers of specific health risks leading to better diagnosis and treatments. In this study, we tested 226 Ixodes ricinis ticks from Southern Germany using a broad-range PCR and electrospray ionization mass spectrometry assay (PCR/ESI-MS) designed to identify tick-borne bacterial and protozoan pathogens in a single test. We found 21.2% of the ticks tested carried Borrelia burgdorferi sensu lato consisting of diverse genospecies; a surprisingly high percentage of ticks were infected with Babesia microti (3.5%). Other organisms found included Borrelia miyamotoi, Rickettsia helvetica, Rickettsia monacensis, and Anaplasma phagocytophilum. Of further significance was our finding that more than 7% of ticks were infected with more than one pathogen or putative pathogen.

Asthma is the most common chronic disease in childhood. Although several therapeutic options are currently available to control the symptoms, many drugs have significant side effects and asthma remains an incurable disease. Microbial exposure in early life reduces the risk of asthma and several studies have suggested protective effects of Toll-like receptor (TLR) activation. We showed previously that modified mRNA provides a safe and efficient therapeutic tool for in vivo gene supplementation. Since current asthma drugs do not take patient specific immune and TLR backgrounds into consideration, treatment with tailored mRNA could be an attractive approach to account for the patient’s individual asthma phenotype. Therefore, we investigated the effect of a preventative treatment with combinations of Tlr1, Tlr2 and Tlr6 mRNA in a House Dust Mite-induced mouse model of asthma. We used chemically modified mRNA which is–in contrast to conventional viral vectors–non-integrating and highly efficient in gene transfer. In our study, we found that treatment with either Tlr1/2 mRNA or Tlr2/6 mRNA, but not Tlr2 mRNA alone, resulted in better lung function as well as reduced airway inflammation in vivo. The present results point to a potentially protective effect of TLR heterodimers in asthma pathogenesis.

BackgroundPhosphoinositide 3-kinase γ (PI3Kγ) and PI3Kδ are second messenger-generating enzymes with key roles in proliferation, differentiation, survival, and function of leukocytes. Deficiency of the catalytic subunits p110γ and p110δ of PI3Kγ and PI3Kδ in p110γ/δ−/− mice leads to defective B- and T-cell homeostasis. Here we examined the role of p110γ and p110δ in the homeostasis of neutrophils by analyzing p110γ−/−, p110δ−/− and p110γ/δ−/− mice.MethodsNeutrophils and T cells in leukocyte suspensions from the bone marrow (BM), blood, spleen and lung were analyzed by flow cytometry. Serum concentrations of IL-17, of the neutrophilic growth factor G-CSF, and of the neutrophil mobilizing CXC chemokines CXCL1/KC and CXCL2/MIP-2 were measured by Bio-Plex assay. Production of G-CSF and CXCL1/KC by IL-17-stimulated primary lung tissue cells were determined by ELISA, whereas IL-17-dependent signaling in lung tissue cells was analyzed by measuring Akt phosphorylation using immunoblot.ResultsWe found that in contrast to single knock-out mice, p110γ/δ−/− mice exhibited significantly elevated neutrophil counts in blood, spleen, and lung. Increased granulocytic differentiation stages in the bone marrow of p110γ/δ−/− mice were paralleled by increased serum concentrations of G-CSF, CXCL1/KC, and CXCL2/MIP-2. As IL-17 induces neutrophilia via the induction of G-CSF and CXC chemokines, we measured IL-17 and IL-17-producing T cells. IL-17 serum concentrations and frequencies of IL-17+ splenic T cells were significantly increased in p110γ/δ−/− mice. Moreover, IFN-γ+, IL-4+, and IL-5+ T cell subsets were drastically increased in p110γ/δ−/− mice, suggesting that IL-17+ T cells were up-regulated in the context of a general percentage increase of other cytokine producing T cell subsets.ConclusionsWe found that p110γ/δ deficiency in mice induces complex immunological changes, which might in concert contribute to neutrophilia. These findings emphasize a crucial but indirect role of both p110γ and p110δ in the regulation of neutrophil homeostasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-017-0185-y) contains supplementary material, which is available to authorized users.

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