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The genome of a second Drosophila species, Drosophila pseudoobscura, is sequenced and is compared to the genome sequence of Drosophila melanogaster, a primary model organism. A repetitive sequence is found in the Drosophila pseudoobscura genome at many junctions between adjacent syntentic blocks.

Huntington disease (HD) is one of a growing number of rare genetic diseases characterized by the inheritance of an increased number of short tandem repeats within the affected gene. Many of these repeat expansion disorders (REDs) affect the brain. Although inheritance of the mutant allele is a necessary first step for disease manifestation, a second step of further expansion of the inherited expanded repeats, particularly in neurons in the brain, also appears to play a critical role toward disease manifestation. This dynamic process, called somatic expansion, is modulated by genes involved in the repair of DNA that operate upstream of the specific gene associated with each individual disease. As somatic expansion has been described in multiple REDs, genes associated with regulating somatic expansion are attractive therapeutic targets, since multiple REDs could potentially be treated with the same drug.

Advancement of RNAi-based therapeutics depends on effective delivery to the site of protein synthesis. Although intravenously administered, multi-component delivery vehicles have enabled small interfering RNA (siRNA) delivery and progression into clinical development, advances of single-component, systemic siRNA delivery have been challenging. In pre-clinical models, attachment of a triantennary N-acetylgalactosamine (GalNAc) ligand to an siRNA mediates hepatocyte uptake via the asialoglycoprotein receptor enabling RNAi-mediated gene silencing. In this phase 1 study, we assessed translation of this delivery approach by evaluating the safety, tolerability, pharmacokinetics, and pharmacodynamics of a GalNAc-siRNA conjugate, revusiran, targeting transthyretin (TTR). Subjects received a placebo or ascending doses of revusiran subcutaneously ranging from 1.25–10 mg/kg in the single and 2.5–10 mg/kg in the multiple ascending dose phases. Revusiran was generally well tolerated, with transient, mild to moderate injection site reactions the most common treatment-emergent adverse events. Doses of 2.5–10 mg/kg revusiran elicited a significant reduction of serum TTR versus the placebo (p <0.01), with mean TTR reductions of approximately 90% observed with multiple dosing. These results demonstrate translation of this novel delivery platform, enabling clinical development of subcutaneously administered GalNAc-siRNAs for liver-based diseases.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to LDL receptors, leading to their degradation. Genetics studies have shown that loss-of-function mutations in PCSK9result in reduced plasma LDL cholesterol and decreased risk of coronary heart disease. We aimed to investigate the safety and efficacy of ALN-PCS, a small interfering RNA that inhibits PCSK9 synthesis, in healthy volunteers with raised cholesterol who were not on lipid-lowering treatment.

Mutations in HFE lead to hereditary hemochromatosis (HH) because of inappropriately high iron uptake from the diet resulting from decreased hepatic expression of the iron-regulatory hormone hepcidin. β-thalassemia is a congenital anemia caused by partial or complete loss of β-globin synthesis causing ineffective erythropoiesis, anemia, decreased hepcidin production, and secondary iron overload. Tmprss6 is postulated to regulate hepcidin production by cleaving Hemojuvelin (Hjv), a key modulator of hepcidin expression, from the hepatocyte surface. On this basis, we hypothesized that treatment of mouse models of HH (Hfe−/−) and β-thalassemia intermedia (Hbbth3/+) with Tmprss6 siRNA formulated in lipid nanoparticles (LNPs) that are preferentially taken up by the liver would increase hepcidin expression and lessen the iron loading in both models. In the present study, we demonstrate that LNP-Tmprss6 siRNA treatment of Hfe−/− and Hbbth3/+ mice induces hepcidin and diminishes tissue and serum iron levels. Furthermore, LNP-Tmprss6 siRNA treatment of Hbbth3/+ mice substantially improved the anemia by altering RBC survival and ineffective erythropoiesis. Our results indicate that pharmacologic manipulation of Tmprss6 with RNAi therapeutics isa practical approach to treating iron overload diseases associated with diminished hepcidin expression and may have efficacy in modifying disease-associated morbidities of β-thalassemia intermedia.

Mutations in HFElead to hereditary hemochromatosis (HH) because of inappropriately high iron uptake from the diet resulting from decreased hepatic expression of the iron-regulatory hormone hepcidin. β-thalassemia is a congenital anemia caused by partial or complete loss of β-globin synthesis causing ineffective erythropoiesis, anemia, decreased hepcidin production, and secondary iron overload. Tmprss6 is postulated to regulate hepcidin production by cleaving Hemojuvelin (Hjv), a key modulator of hepcidin expression, from the hepatocyte surface. On this basis, we hypothesized that treatment of mouse models of HH (Hfe−/−) and β-thalassemia intermedia (Hbbth3/+) with Tmprss6 siRNA formulated in lipid nanoparticles (LNPs) that are preferentially taken up by the liver would increase hepcidin expression and lessen the iron loading in both models. In the present study, we demonstrate that LNP-Tmprss6 siRNA treatment of Hfe−/−and Hbbth3/+mice induces hepcidin and diminishes tissue and serum iron levels. Furthermore, LNP-Tmprss6 siRNA treatment of Hbbth3/+mice substantially improved the anemia by altering RBC survival and ineffective erythropoiesis. Our results indicate that pharmacologic manipulation of Tmprss6 with RNAi therapeutics isa practical approach to treating iron overload diseases associated with diminished hepcidin expression and may have efficacy in modifying disease-associated morbidities of β-thalassemia intermedia.

Lck, one of eight members of the Src family of tyrosine kinases, is activated after T cell stimulation and is required for T-cell proliferation and interleukin (IL)-2 production. Inhibition of Lck has been a target to prevent lymphocyte activation and acute rejection. Here, we report the pharmacologic characterization of 1-methyl-1 H-indole-2-carboxylic acid (4-{1-[4-(4-acetyl-piperazin-l-yl)-cyclohexyl]-4-amino-1 H-pyrazolo[3,4-d]pyrimidin-3-yl}-2-methoxy-phenyl)-amide (A-770041), an orally bioavailable pyrazolo[3,4-d]pyrimidine with increased selectivity for Lck compared with previously reported compounds. A-770041 is a 147 nM inhibitor of Lck (1 mM ATP) and is 300-fold selective against Fyn, the other Src family kinase involved in T-cell signaling. Concanavalin A-stimulated IL-2 production in whole blood is inhibited by A-770041 with an EC50of approximately 80 nM. A-770041 is orally bioavailable (F= 34.1 ± 7.2% at 10 mg/kg) and has a t1/2of 4.1 ± 0.1 h. Concanavalin A-induced IL-2 production in vivo is inhibited by oral administration of A-770041 (in vivo EC50= 78 ± 28 nM). Doses of A-770041 at or above 10 mg/kg/day prevent rejection of hearts transplanted heterotopically in rats from Brown Norway donors to Lewis recipients across a major histocompatibility barrier for least 65 days. Grafts from animals treated with 20 mg/kg/day A-770041 or 10 mg/day Cyclosporin A had minimal microvascular changes or multifocal mononuclear infiltrates. However, mineralization in myocytes from the grafts from A-770041-treated animals was less than animals treated with Cyclosporin A. Lck inhibition is an attractive target to prevent acute rejection.

We recently showed that a soluble, heterodimeric murine D10 T-cell receptor (TCR) (Vα2Cα, Vβ8.2Cβ) expressed in insect cells binds both Vβ8.2-specific bacterial superantigen staphylococcal enterotoxin C2 (SEC2) and a soluble, heterodimeric major histocompatibility complex class II I-Ak·conalbumin peptide complex with a low micromolar affinity. To define further the structural requirements for the TCR/ligand interactions, we have produced in Escherichia colia soluble, functional D10 single chain (sc) TCR molecule in which the Vα and Vβ domains are connected by a flexible peptide linker. Purified and refolded D10 scTCR bound to SEC2 and murine major histocompatibility complex class II I-Ak·conalbumin peptide complex with thermodynamic and kinetic binding constants similar to those measured for the baculovirus-derived heterodimeric D10 TCR suggesting that neither the TCR constant domains nor potential N- or O-linked carbohydrate moieties are necessary for ligand recognition and for expression and proper folding of the D10 scTCR. Purified D10 scTCR remained soluble at concentrations up to 1 mm. Circular dichroism and NMR spectroscopy indicated that D10 scTCR is stabilized predominantly by β-sheet secondary structure, consistent with its native-like conformation. Because of its limited size, high solubility, and structural integrity, purified D10 scTCR appears to be suitable for structural studies by multidimensional NMR spectroscopy.

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The b-Thalassemias are a group of hereditary blood disorders resulting from insufficient beta globin production, ultimately giving rise to the signature clinical sequelae associated with β-Thalassemia which includes anemia, ineffective erythropoiesis, and secondary iron overload. Previously, we demonstrated that intravenous administration of an siRNA targeting hepatic Tmprss6expression significantly ameliorated the disease phenotype in the Hbbth3/+mouse model of Thalassemia Intermedia (Blood.2013; 14;121(7):1200-8). The Tmprss6gene encodes for the protein Matriptase-2 which negatively regulates Hepcidin gene expression by cleaving the Hepcidin regulatory protein Hemojuvelin; RNAi-mediated suppression of Tmprss6removes this negative regulator, ultimately leading to an increase in Hepcidin expression. Increased Hepcidin expression leads to a significant decrease in serum iron concentration and Transferrin Saturation (TfSat), which in the β-Thalassemia disease setting, corrects ineffective erythropoiesis, ameliorates anemia, and mitigates secondary iron overload. The role of Tmprss6in iron metabolism has been extensively characterized in animal and human studies and, together with the observation in the Hbbth3/+mouse, represents an attractive therapeutic target for the treatment of β-Thalassemia.

Toudjarska: Alnylam Pharmaceuticals, Inc.: Employment. Cai:Alnylam Pharmaceuticals, Inc.: Employment. Racie:Alnylam Pharmaceuticals, Inc.: Employment. Milstein:Alnylam Pharmaceuticals, Inc.: Employment. Bettencourt:Alnylam Pharmaceuticals, Inc.: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Sah:Alnylam Pharmaceuticals, Inc.: Employment. Vaishnaw:Alnylam Pharmaceuticals, Inc.: Employment. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment.

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Toudjarska: Alnylam Pharmaceuticals, Inc.: Employment. Cai:Alnylam Pharmaceuticals, Inc.: Employment. Racie:Alnylam Pharmaceuticals, Inc.: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Milstein:Alnylam Pharmaceuticals, Inc.: Employment. Bettencourt:Alnylam Pharmaceuticals, Inc.: Employment. Sah:Alnylam Pharmaceuticals, Inc.: Employment. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment. Maraganore:Alnylam pharmaceuticals, Inc.: Employment. Vaishnaw:Alnylam Pharmaceuticals, Inc.: Employment.

Abstract 1045

Background: The recent completion of the Drosophila melanogastergenomic sequence to high quality and the availability of a greatly expanded set of DrosophilacDNA sequences, aligning to 78% of the predicted euchromatic genes, afforded FlyBase the opportunity to significantly improve genomic annotations. We made the annotation process more rigorous by inspecting each gene visually, utilizing a comprehensive set of curation rules, requiring traceable evidence for each gene model, and comparing each predicted peptide to SWISS-PROT and TrEMBL sequences. Results: Although the number of predicted protein-coding genes in Drosophilaremains essentially unchanged, the revised annotation significantly improves gene models, resulting in structural changes to 85% of the transcripts and 45% of the predicted proteins. We annotated transposable elements and non-protein-coding RNAs as new features, and extended the annotation of untranslated (UTR) sequences and alternative transcripts to include more than 70% and 20% of genes, respectively. Finally, cDNA sequence provided evidence for dicistronic transcripts, neighboring genes with overlapping UTRs on the same DNA sequence strand, alternatively spliced genes that encode distinct, non-overlapping peptides, and numerous nested genes. Conclusions: Identification of so many unusual gene models not only suggests that some mechanisms for gene regulation are more prevalent than previously believed, but also underscores the complex challenges of eukaryotic gene prediction. At present, experimental data and human curation remain essential to generate high-quality genome annotations.

Low to moderate levels of stress induce a class of molecular chaperones called heat shock proteins (Hsps), which protect cells, tissues and whole organisms from more severe stress. In higher Eukaryotes, Hsp70 is one of the principle heat-induced chaperones. This response is general, and how much Hsp70 an animal produces correlates with the level of stress to which it is exposed. Nonetheless, definitively linking high Hsp70 expression as an adaptation to stress tolerance is problematic, because organisms and cells respond to stress in many ways. By molecular manipulation of Hsp70 in one animal group, Drosophila, differences in hsp70 copy number are shown to directly influence heat-induced expression of Hsp70 and tolerance of heat. However, too high an expression level of Hsp70 can harm individuals during periods of rapid growth. This strong physiological relationship between Hsp70 concentration and thermotolerance, along with Hsp70's remarkable degree of interspecific coding sequence conservation, suggest that hsp70 regulatory elements may evolve as an adaptation in diverse species to their thermal environments. To examine this possibility, correlative studies within species and research on phylogenetic covariation between these traits is reviewed with a focus on Drosophila species. However, the techniques and results discussed should broadly apply to other animal groups where evolutionary approaches can be used to test whether genetic variation in both thermotolerance and Hsp expression within and among species select locally on either hsp70 sequence and/or expression.