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An siRNA targeting antithrombin promotes hemostasis in mouse and nonhuman primate models of hemophilia and could represent a new therapeutic option for this disease. Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.

In vivo silencing in specific cell types remains the main obstacle for therapeutic applications of siRNAs. Leuschner et al . now show that an optimized lipid nanoparticle delivers siRNA to inflammatory monocytes in mice and, when transporting CCR2 siRNA, has therapeutic effects in cardiovascular disease, cancer and transplant rejection. Excessive and prolonged activity of inflammatory monocytes is a hallmark of many diseases with an inflammatory component. In such conditions, precise targeting of these cells could be therapeutically beneficial while sparing many essential functions of the innate immune system, thus limiting unwanted effects. Inflammatory monocytes—but not the noninflammatory subset—depend on the chemokine receptor CCR2 for localization to injured tissue. Here we present an optimized lipid nanoparticle and a CCR2-silencing short interfering RNA that, when administered systemically in mice, show rapid blood clearance, accumulate in spleen and bone marrow, and localize to monocytes. Efficient degradation of CCR2 mRNA in monocytes prevents their accumulation in sites of inflammation. Specifically, the treatment attenuates their number in atherosclerotic plaques, reduces infarct size after coronary artery occlusion, prolongs normoglycemia in diabetic mice after pancreatic islet transplantation, and results in reduced tumor volumes and lower numbers of tumor-associated macrophages.

The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology.

Cellular functions are mediated through complex systems of macromolecules and metabolites linked through biochemical and physical interactions, represented in interactome models as ‘nodes’ and ‘edges’, respectively. Better understanding of genotype‐to‐phenotype relationships in human disease will require modeling of how disease‐causing mutations affect systems or interactome properties. Here we investigate how perturbations of interactome networks may differ between complete loss of gene products (‘node removal’) and interaction‐specific or edge‐specific (‘edgetic’) alterations. Global computational analyses of ∼50 000 known causative mutations in human Mendelian disorders revealed clear separations of mutations probably corresponding to those of node removal versus edgetic perturbations. Experimental characterization of mutant alleles in various disorders identified diverse edgetic interaction profiles of mutant proteins, which correlated with distinct structural properties of disease proteins and disease mechanisms. Edgetic perturbations seem to confer distinct functional consequences from node removal because a large fraction of cases in which a single gene is linked to multiple disorders can be modeled by distinguishing edgetic network perturbations. Edgetic network perturbation models might improve both the understanding of dissemination of disease alleles in human populations and the development of molecular therapeutic strategies. Synopsis Genotype‐to‐phenotype relationships in human genetic disease are often modeled as: ‘mutation in gene X leads to loss of gene product X, which leads to disease A’. However, single ‘gene‐loss’ models cannot explain the increasingly appreciated prevalence of complex genotype‐to‐phenotype relationships, particularly with instances of allelic or locus hetrogeneity (Goh et al , 2007 ). Genes and gene products function not in isolation but as components of complex networks of macromolecules (DNA, RNA, or proteins) and metabolites linked through biochemical or physical interactions, often represented in ‘interactome’ network models as ‘nodes’ and ‘edges’, respectively. Here we use network perturbation models to explain molecular dysfunctions underlying human disease in addition to the gene‐loss model. We hypothesize that different mutations leading to different molecular defects to proteins may cause distinct perturbations of cellular networks, giving rise to distinct phenotypic outcomes (Figure 1 ). For example, truncations close to the start of an open‐reading frame, or mutations that grossly destabilize a protein structure, can be modeled as removing a protein node from the network (‘node removal’). Alternatively, single amino‐acid substitutions that affect specific binding sites, or truncations that preserve certain domains of a protein, may give rise to partially functional gene products with specific changes in distinct molecular interaction(s) (edge‐specific or ‘edgetic’ perturbations) (Figure 1B ). Taking advantage of the large number of known disease‐causing allelic variations in human Mendelian disorders, we investigated how disease‐associated mutations may cause complete loss of gene products or, alternatively, may cause specific loss or gain of individual molecular interaction(s). We examined ∼50 000 Mendelian disease‐causing alleles, affecting over 1900 protein‐coding genes, altogether associated with more than 2000 human disorders available in the Human Gene Mutation Database (HGMD) (Stenson et al , 2003 ), that can be subdivided into two subsets: truncating’ alleles (truncations or frameshifts caused by stop codons, out‐of‐frame insertions or deletions, or defective splicing) versus ‘in‐frame’ alleles (missense mutations and in‐frame insertions or deletions). Over 50% (27 919/52 491) of Mendelian alleles in HGMD correspond to ‘in‐frame’ mutations. Our hypothesis is that, ‘in‐frame’ alleles may affect specific interactions of a given gene product while leaving most other interactions unperturbed. Although exceptions may apply, our hypothesis has several predictions. First, ‘truncating’ versus ‘in‐frame’ alleles may distribute differently among autosomal dominant and autosomal recessive disease, given that dominant mutations are more likely to be edgetic than recessive ones. Indeed, autosomal dominant and autosomal recessive traits annotated in the Online Mendelian Inheritance in Man (OMIM) database (Hamosh et al , 2005 ) show a clear separation with respect to the associated ‘in‐frame’ versus ‘truncating’ mutations. Among genes affected solely by ‘in‐frame’ mutations, the proportion of dominant diseases is ∼10‐fold higher than that of recessive ones, supporting ‘in‐frame’ mutations causing distinct molecular defects as opposed to ‘truncating’ mutations. A proof‐of‐principle characterization of binary protein interaction defects of mutant alleles associated with five genetic disorders supports our hypothesis that ‘in‐frame’ alleles indeed produce mostly functional proteins, preserving many specific protein interactions. As grossly disruptive mutations versus mutations leading to loss or gain of specific interaction(s) probably distribute differently on protein structures, we examined available three‐dimensional structures of all disease proteins. Mutated residues in autosomal dominant disease are significantly more exposed to the surface of the structure than those in autosomal recessive disease, consistent with the idea that disease with distinct modes of inheritance probably involves distinct network perturbations. A second testable prediction of our edgetic perturbation model is that edgetic perturbation versus gene loss for a given gene product might in some cases cause different diseases. We examined 142 genes associated with two or more distinct diseases in which at least five distinct alleles have been reported for each disease. We found ∼30% of the cases for which distribution of ‘in‐frame’ versus ‘truncating’ mutations is significantly different between the two diseases linked to the same gene ( P <0.05). Hence, when affecting the same gene, node removal versus edgetic perturbation can confer strikingly different phenotypes. A third testable prediction is that different edgetic perturbations for a given gene product might cause phenotypically distinguishable diseases (Figure 6 ). We used predicted Pfam domains (Finn et al , 2006 ) as surrogates for functional interaction domains, assuming that ‘in‐frame’ mutations located in distinct Pfam domain‐encoding sequences probably alter distinct interactions. Among 169 genes associated with two or more diseases and encoding proteins containing at least two Pfam domains, nine proteins have at least two Pfam domains significantly enriched with ‘in‐frame’ mutations ( P <0.05). For each of the nine proteins, we found a striking pattern of near mutual exclusivity, whereby different Pfam domains seem to be specifically affected in distinct disorders (Figure 6B ). We conclude that edgetic alleles probably underlie many complex genotype‐to‐phenotype relationships in human disease, such as incomplete penetrance or variable expressivity, as well as allele‐specific phenotypic variations among patients. Edgetic perturbation of human inherited disorders might help explain how seemingly devastating alleles have appeared and persevered in human populations. We present alternative models to explain molecular dysfunctions underlying human inherited disorders based on interaction‐specific or “edgetic” perturbations rather than complete loss of gene products. We find that a substantial fraction of known genetic variants in human Mendelian disorders likely cause edgetic perturbations. We find frequent situations where edgetic perturbation models can explain how different mutations in a single gene can cause distinct disorders. Edgetic perturbation models should provide alternative explanations to complex genotype‐to‐phenotype relationships

High-throughput yeast two-hybrid screening is used to generate the largest C. elegans interactome resource available thus far. Using an empirical quality control framework presented in Venkatesan et al ., also online, the data set is evaluated for quality and is used to estimate the total size of the worm interactome. To provide accurate biological hypotheses and elucidate global properties of cellular networks, systematic identification of protein-protein interactions must meet high quality standards. We present an expanded C. elegans protein-protein interaction network, or 'interactome' map, derived from testing a matrix of ∼10,000 × ∼10,000 proteins using a highly specific, high-throughput yeast two-hybrid system. Through a new empirical quality control framework, we show that the resulting data set (Worm Interactome 2007, or WI-2007) was similar in quality to low-throughput data curated from the literature. We filtered previous interaction data sets and integrated them with WI-2007 to generate a high-confidence consolidated map (Worm Interactome version 8, or WI8). This work allowed us to estimate the size of the worm interactome at ∼116,000 interactions. Comparison with other types of functional genomic data shows the complementarity of distinct experimental approaches in predicting different functional relationships between genes or proteins.

Recent GWAS have identified SNPs at a human chromosom1 locus associated with coronary artery disease risk and LDL cholesterol levels. The SNPs are also associated with altered expression of hepatic sortilin-1 (SORT1), which encodes a protein thought to be involved in apoB trafficking and degradation. Here, we investigated the regulation of Sort1 expression in mouse models of obesity. Sort1 expression was markedly repressed in both genetic (ob/ob) and high-fat diet models of obesity; restoration of hepatic sortilin-1 levels resulted in reduced triglyceride and apoB secretion. Mouse models of obesity also exhibit increased hepatic activity of mammalian target of rapamycin complex 1 (mTORC1) and ER stress, and we found that administration of the mTOR inhibitor rapamycin to ob/ob mice reduced ER stress and increased hepatic sortilin-1 levels. Conversely, genetically increased hepatic mTORC1 activity was associated with repressed Sort1 and increased apoB secretion. Treating WT mice with the ER stressor tunicamycin led to marked repression of hepatic sortilin-1 expression, while administration of the chemical chaperone PBA to ob/ob mice led to amelioration of ER stress, increased sortilin-1 expression, and reduced apoB and triglyceride secretion. Moreover, the ER stress target Atf3 acted at the SORT1 promoter region as a transcriptional repressor, whereas knockdown of Atf3 mRNA in ob/ob mice led to increased hepatic sortilin-1 levels and decreased apoB and triglyceride secretion. Thus, in mouse models of obesity, induction of mTORC1 and ER stress led to repression of hepatic Sort1 and increased VLDL secretion via Atf3. This pathway may contribute to dyslipidemia in metabolic disease.

Cytoplasmic polyadenylation element binding (CPEB) proteins are evolutionary conserved RNA-binding proteins that control mRNA polyadenylation and translation. Orthologs in humans and other vertebrates are mainly involved in oogenesis. This is also the case for the C. elegans CPEB family member CPB-3, whereas two further CPEB proteins (CPB-1 and FOG-1) are involved in spermatogenesis. Here we describe the characterisation of a new missense allele of cpb-3 and show that loss of cpb-3 function leads to an increase in physiological germ cell death. To better understand the interaction and effect of C. elegans CPEB proteins on processes such as physiological apoptosis, germ cell differentiation, and regulation of gene expression, we characterised changes in the transcriptome and proteome of C. elegans CPEB mutants. Our results show that, despite their sequence similarities CPEB family members tend to have distinct overall effects on gene expression (both at the transcript and protein levels). This observation is consistent with the distinct phenotypes observed in the various CPEB family mutants.

A gene conferring neomycin resistance can be used for antibiotic selection in C. elegans and C. briggsae . This will permit easy maintenance of transgenic lines and facilitate single-copy insertion of transgenes. Also in this issue, a related paper reports nematode selection using puromycin. We have developed a nematode transformation vector carrying the bacterial neomycin resistance gene ( NeoR ) and shown that it could confer resistance to G-418 on both wild-type Caenorhabditis elegans and C. briggsae . This selection system allows hands-off maintenance and enrichment of transgenic worms carrying non-integrated transgenes on selective plates. We also show that this marker can be used for Mos1-mediated single-copy insertion in wild-type genetic backgrounds (MosSCI-biotic).

Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.

A combination of forward and reverse two hybrid screening allows systematic identification of 'edgetic' or edge-specific alleles, which encode proteins that have lost a single physical interaction but for which other interactions remain unperturbed. Genes and gene products do not function in isolation but within highly interconnected 'interactome' networks, modeled as graphs of nodes and edges representing macromolecules and interactions between them, respectively. We propose to investigate genotype-phenotype associations by methodical use of alleles that lack single interactions, while retaining all others, in contrast to genetic approaches designed to eliminate gene products completely. We describe an integrated strategy based on the reverse yeast two-hybrid system to isolate and characterize such edge-specific, or 'edgetic', alleles. We established a proof of concept with CED-9, a Caenorhabditis elegans BCL2 ortholog. Using ced-9 edgetic alleles, we uncovered a new potential functional link between apoptosis and a centrosomal protein. This approach is amenable to higher throughput and is particularly applicable to interactome network analysis in organisms for which transgenesis is straightforward.