Background:The inflammasome generates IL-1 family proteins, but its role in neutrophils is poorly understood. Results: Neutrophils store key inflammasome components in distinct intracellular compartments and release IL-1␤ and IL-18, but not IL-1␣ or IL-33. Conclusion: Neutrophils store inflammasome components in intracellular compartments. Significance: Targeting the inflammasome in neutrophils represents a future anti-inflammatory strategy.

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...

Patients with CF who have chronic P. aeruginosa infection show an age-dependent, quantitative, and qualitative impairment of Tregs. Modulation of Tregs represents a novel strategy to rebalance T-cell responses, dampen inflammation, and ultimately improve outcomes for patients with infective CF lung disease.

No abstract