TGF-␤-activated kinase 1 (TAK1),[Keywords: JNK; NF-B; TAB1; TAB2; TAK1; TGF-␤] Supplemental material is available at http://www.genesdev.org.

The ability of small interfering RNA (siRNA) to efficiently silence the expression of specific genes provides the basis for exciting new therapies based on RNA interference (RNAi). The efficient intracellular delivery of siRNA from cell uptake through the endosomal trafficking pathways into the cytoplasm remains a significant challenge. Previously we described the synthesis of a new family of diblock copolymer siRNA carriers using controlled reversible addition fragmentation chain transfer (RAFT) polymerization. The carriers were composed of a positively-charged block of dimethylaminoethyl methacrylate (DMAEMA) to mediate siRNA binding and a second pH-responsive endosome releasing block composed of DMAEMA and propylacrylic acid (PAA) in roughly equimolar ratios, and butyl methacylate (BMA). Here we describe the development of a new generation of siRNA delivery polymers based on this design that exhibit enhanced transfection efficiency and low cytotoxicity. This design incorporates a longer endosomolytic block with increased hydrophobic content to induce micelle formation. These polymers spontaneously form spherical micelles in the size range of 40 nm with CMC (critical micelle concentration) values of approximately 2 μg/ml based on dynamic light scattering (DLS), 1H-NMR, electron microscopy, and selective partitioning of the small molecule pyrene into the hydrophobic micelle core. The siRNA binding to the cationic shell block did not perturb micelle stability or significantly increase particle size. The self-assembly of the diblock copolymers into particles was shown to provide a significant enhancement in mRNA knockdown at siRNA concentrations as low as 12.5 nM. Under these conditions the micelle--based systems showed an 89 % reduction in GAPDH mRNA levels as compared to only 23 % (10 nM siRNA) for the non-micelle system. The reduction in mRNA levels becomes nearly quantitative as the siRNA concentration is increased to 25 nM and higher. Flow cytometry analysis of fluorescent-labeled siRNA showed uptake in 90% of cells and a 3-fold increase in siRNA per cell compared to a standard lipid transfection agent. These results demonstrate the potential utility of this carrier design for siRNA drug delivery.

We describe a novel, two-nanoparticle mRNA delivery system and show that it is highly effective as a means of intracellular enzyme replacement therapy (i-ERT) using a murine model of ornithine transcarbamylase deficiency (OTCD). Our Hybrid mRNA Technology delivery system (HMT) comprises an inert lipid nanoparticle that protects the mRNA from nucleases in the blood as it distributes to the liver and a polymer micelle that targets hepatocytes and triggers endosomal release of mRNA. This results in high-level synthesis of the desired protein specifically in the liver. HMT delivery of human OTC mRNA normalizes plasma ammonia and urinary orotic acid levels, and leads to a prolonged survival benefit in the murine OTCD model. HMT represents a unique, non-viral mRNA delivery method that allows multi-dose, systemic administration for treatment of single-gene inherited metabolic diseases.

Arsenic oxidation is recognized as being mediated by both heterotrophic and chemoautotrophic microorganisms. Enrichment cultures were established to determine whether chemoautotrophic microorganisms capable of oxidizing arsenite As(III) to arsenate As(V) are present in selected contaminated but nonextreme environments. Three new organisms, designated as strains OL-1, S-1 and CL-3, were isolated and found to oxidize 10 mM arsenite to arsenate under aerobic conditions using CO2-bicarbonate (CO2/HCO3-) as a carbon source. Based on 16S rRNA gene sequence analyses, strain OL-1 was 99% most closely related to the genus Ancylobacter, strain S-1 was 99% related to Thiobacillus and strain CL-3 was 98% related to the genus Hydrogenophaga. The isolates are facultative autotrophs and growth of isolated strains on different inorganic electron donors other than arsenite showed that all three had a strong preference for several sulfur species, while CL-3 was also able to grow on ammonium and nitrite. The RuBisCO Type I (cbbL) gene was positively amplified and sequenced in strain CL-3, and the Type II (cbbM) gene was detected in strains OL-1 and S-1, supporting the autotrophic nature of the organisms.