Search

Alpha-1 antitrypsin (AAT) is a serum protease inhibitor that belongs to the serpin superfamily. Mutations in AAT are associated with α-1 antitrypsin deficiency (AATD), a rare genetic disease with two distinct manifestations: AATD lung disease and AATD liver disease. AATD lung disease is caused by loss-of-function of AAT and can be treated with plasma-derived AAT. AATD liver disease is due to the aggregation and retention of mutant AAT protein in the liver; the only treatment available for AATD liver disease is liver transplantation. Here we demonstrate that antisense oligonucleotides (ASOs) targeting human AAT efficiently reduce levels of both short and long human AAT transcript in vitro and in transgenic mice, providing a novel therapy for AATD liver disease. In addition, ASO-mediated depletion of mouse AAT may offer a useful animal model for the investigation of AATD lung disease.

Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.

Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disease that results from mutations in the alpha-1 antitrypsin (AAT) gene. The mutant AAT protein aggregates and accumulates in the liver leading to AATD liver disease, which is only treatable by liver transplant. The PiZ transgenic mouse strain expresses a human AAT (hAAT) transgene that contains the AATD-associated Glu342Lys mutation. PiZ mice exhibit many AATD symptoms, including AAT protein aggregates, increased hepatocyte death, and liver fibrosis. In the present study, we systemically treated PiZ mice with an antisense oligonucleotide targeted against hAAT (AAT-ASO) and found reductions in circulating levels of AAT and both soluble and aggregated AAT protein in the liver. Furthermore, AAT-ASO administration in these animals stopped liver disease progression after short-term treatment, reversed liver disease after long-term treatment, and prevented liver disease in young animals. Additionally, antisense oligonucleotide treatment markedly decreased liver fibrosis in this mouse model. Administration of AAT-ASO in nonhuman primates led to an approximately 80% reduction in levels of circulating normal AAT, demonstrating potential for this approach in higher species. Antisense oligonucleotides thus represent a promising therapy for AATD liver disease.

Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls. Antisense oligonucleotides (ASOs) are identified that reduce GGGGCC-containing nuclear foci without altering overall C9orf72 RNA levels. By contrast, siRNAs fail to reduce nuclear RNA foci despite marked reduction in overall C9orf72 RNAs. Sustained ASO-mediated lowering of C9orf72 RNAs throughout the CNS of mice is demonstrated to be well tolerated, producing no behavioral or pathological features characteristic of ALS/FTD and only limited RNA expression alterations. Genome-wide RNA profiling identifies an RNA signature in fibroblasts from patients with C9orf72 expansion. ASOs targeting sense strand repeat-containing RNAs do not correct this signature, a failure that may be explained, at least in part, by discovery of abundant RNA foci with C9orf72 repeats transcribed in the antisense (GGCCCC) direction, which are not affected by sense strand-targeting ASOs. Taken together, these findings support a therapeutic approach by ASO administration to reduce hexanucleotide repeat-containing RNAs and raise the potential importance of targeting expanded RNAs transcribed in both directions.

The NMOSFET-only pass gates used in some digital CMOS applications, such as the Field-Programmable Gate Arrays (FPGAs), are apparently vulnerable to Positive Bias Temperature Instability (PBTI). Here we discuss the impact of PBTI frequency and workload on high-k/metal-gate NMOSFETs in terms of Capture-and-Emission Time (CET) maps and quantitatively explain the degradation of our test circuit. From individual trapping events in deeply-scaled NMOSFETs we then project PBTI distributions at 10 years. Finally, we show that at increased supply voltage the pass gate speed degradation is outweighed by signal transfer speedup, resulting in a net performance improvement.

Autosomal dominant diseases such as Huntington's disease (HD) are caused by a gain of function mutant protein and/or RNA. An ideal treatment for these diseases is to selectively suppress expression of the mutant allele while preserving expression of the wild-type variant. RNase H active antisense oligonucleotides (ASOs) or small interfering RNAs can achieve allele selective suppression of gene expression by targeting single nucleotide polymorphisms (SNPs) associated with the repeat expansion. ASOs have been previously shown to discriminate single nucleotide changes in targeted RNAs with ∼5-fold selectivity. Based on RNase H enzymology, we enhanced single nucleotide discrimination by positional incorporation of chemical modifications within the oligonucleotide to limit RNase H cleavage of the non-targeted transcript. The resulting oligonucleotides demonstrate >100-fold discrimination for a single nucleotide change at an SNP site in the disease causing huntingtin mRNA, in patient cells and in a completely humanized mouse model of HD. The modified ASOs were also well tolerated after injection into the central nervous system of wild-type animals, suggesting that their tolerability profile is suitable for advancement as potential allele-selective HD therapeutics. Our findings lay the foundation for efficient allele-selective downregulation of gene expression using ASOs-an outcome with broad application to HD and other dominant genetic disorders.

In this work, the impact of bias temperature instability is evaluated for routing pass gate circuits. A simple test structure is proposed and measured data is compared to aging models to demonstrate the importance of modeling circuit level aging effects. Aging models which are shown to be accurate at the transistor level are inadequate at the circuit level unless frequency dependent aging effects are taken into account.

Background: Prion protein (PrPC) is a receptor for amyloid-β oligomers (AβOs). Results: AβO binding to PrPC and cytotoxicity require transmembrane LRP1 and are reduced by cholesterol depletion and AβO remodeling. Conclusion: Cellular binding and toxicity of AβOs are dependent on PrPC being localized in a raft-based complex. Significance: Remodeling AβOs and disrupting the prion-LRP1-raft interaction provide therapeutic targets for Alzheimer disease., Soluble oligomers of the amyloid-β (Aβ) peptide cause neurotoxicity, synaptic dysfunction, and memory impairments that underlie Alzheimer disease (AD). The cellular prion protein (PrPC) was recently identified as a high affinity neuronal receptor for Aβ oligomers. We report that fibrillar Aβ oligomers recognized by the OC antibody, which have been shown to correlate with the onset and severity of AD, bind preferentially to cells and neurons expressing PrPC. The binding of Aβ oligomers to cell surface PrPC, as well as their downstream activation of Fyn kinase, was dependent on the integrity of cholesterol-rich lipid rafts. In SH-SY5Y cells, fluorescence microscopy and co-localization with subcellular markers revealed that the Aβ oligomers co-internalized with PrPC, accumulated in endosomes, and subsequently trafficked to lysosomes. The cell surface binding, internalization, and downstream toxicity of Aβ oligomers was dependent on the transmembrane low density lipoprotein receptor-related protein-1 (LRP1). The binding of Aβ oligomers to cell surface PrPC impaired its ability to inhibit the activity of the β-secretase BACE1, which cleaves the amyloid precursor protein to produce Aβ. The green tea polyphenol (−)-epigallocatechin gallate and the red wine extract resveratrol both remodeled the fibrillar conformation of Aβ oligomers. The resulting nonfibrillar oligomers displayed significantly reduced binding to PrPC-expressing cells and were no longer cytotoxic. These data indicate that soluble, fibrillar Aβ oligomers bind to PrPC in a conformation-dependent manner and require the integrity of lipid rafts and the transmembrane LRP1 for their cytotoxicity, thus revealing potential targets to alleviate the neurotoxic properties of Aβ oligomers in AD.

FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from >5,500 genes in mouse and human brain, primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern, consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of >950 mRNAs, most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain, but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell-derived human neurons and in TDP-43 aggregate-containing motor neurons in sporadic ALS, supporting a common loss-of-function pathway as one component underlying motor neuron death from misregulation of TDP-43 or FUS/TLS.

RNA processing is vital for the high fidelity and diversity of eukaryotic transcriptomes and the encoded proteomes. However, control of RNA processing is not fully established. Σ RNA is a class of conserved large non-coding RNAs (murine Hepcarcin; human MALAT-1) up-regulated in carcinomas. Using antisense technology, we identified that RNA post-transcriptional modification is the most significant global function of Σ RNA. Specifically, processing of the pre-mRNAs of genes including Tissue Factor and Endoglin was altered by hydrolysis of Σ RNA/MALAT-1. These results support the hypothesis that Σ RNA/MALAT-1 is a regulatory molecule exerting roles in RNA post-transcriptional modification.

Zinc, the most abundant trace metal in the brain, has numerous functions, both in health and in disease. Zinc is released into the synaptic cleft of glutamatergic neurons alongside glutamate from where it interacts and modulates NMDA and AMPA receptors. In addition, zinc has multifactorial functions in Alzheimer's disease (AD). Zinc is critical in the enzymatic nonamyloidogenic processing of the amyloid precursor protein (APP) and in the enzymatic degradation of the amyloid-β(Aβ) peptide. Zinc binds to Aβpromoting its aggregation into neurotoxic species, and disruption of zinc homeostasis in the brain results in synaptic and memory deficits. Thus, zinc dyshomeostasis may have a critical role to play in the pathogenesis of AD, and the chelation of zinc is a potential therapeutic approach.

Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the AS of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight the role for an nrRNA in the regulation of gene expression.

The impact of process local variation on FPGA configuration memory is studied in this paper. Memory cell stability is examined by simulations and experiments on 65nm and 45nm processes. A statistical simulation method, which correlates closely with product silicon, has been developed. The results show that the trend of process local variation and memory density scaling adversely impact FPGA configuration memory stability. It is found that the cell stability is greatly affected by cell layout.

We give a case-free proof that the lattice of noncrossing partitions associated to any finite real reflection group is EL-shellable. Shellability of these lattices was open for the groups of type Dn and those of exceptional type and rank at least three. © 2006 American Mathematical Society.

The cellular prion protein (PrPC) is essential for the pathogenesis and transmission of prion diseases. Although PrPC is known to be located in detergent-insoluble lipid rafts at the surface of neuronal cells, the mechanism of its internalisation is unclear, with both raft/caveolae-based and clathrin-mediated processes being proposed. We have investigated the mechanism of copper-induced internalisation of PrPC in neuronal cells by immunofluorescence microscopy, surface biotinylation assays and buoyant sucrose density gradient centrifugation in the presence of Triton X-100. Clathrin-mediated endocytosis was selectively blocked with tyrphostin A23, which disrupts the interaction between tyrosine motifs in the cytosolic domains of integral membrane proteins and the adaptor complex AP2, and a dominant-negative mutant of the adaptor protein AP180. Both these agents inhibited the copper-induced endocytosis of PrPC. Copper caused PrPC to move laterally out of detergent-insoluble lipid rafts into detergent-soluble regions of the plasma membrane. Using mutants of PrPC that lack either the octapeptide repeats or the N-terminal polybasic region, and a construct with a transmembrane anchor, we show that copper binding to the octapeptide repeats promotes dissociation of PrPC from lipid rafts, whereas the N-terminal polybasic region mediates its interaction with a transmembrane adaptor protein that engages the clathrin endocytic machinery. Our results provide an experimental basis for reconciling the apparently contradictory observations that the prion protein undergoes clathrin-dependent endocytosis despite being localised in lipid rafts. In addition, we have been able to assign distinct functions in the endocytic process to separate regions of the protein.

The cellular prion protein (PrP(C)) is critical for the development of prion diseases. However, the physiological role of PrP(C) is less clear, although a role in the cellular resistance to oxidative stress has been proposed. PrP(C) is cleaved at the end of the copper-binding octapeptide repeats through the action of reactive oxygen species (ROS), a process termed beta-cleavage. Here we show that ROS-mediated beta-cleavage of cell surface PrP(C) occurs within minutes and was inhibited by the hydroxyl radical quencher dimethyl sulfoxide and by an antibody against the octapeptide repeats. A construct of PrP lacking the octapeptide repeats, PrPDeltaoct, failed to undergo ROS-mediated beta-cleavage, as did two mutant forms of PrP, PG14 and A116V, associated with human prion diseases. As compared with cells expressing wild type PrP, when challenged with H2O2 and Cu2+, cells expressing PrPdeltaoct, PG14, or A116V had reduced viability and glutathione peroxidase activity and increased intracellular free radicals. Thus, lack of ROS-mediated beta-cleavage of PrP correlated with the sensitivity of the cells to oxidative stress. These data indicate that the beta-cleavage of PrP(C) is an early and critical event in the mechanism by which PrP protects cells against oxidative stress.

The U.S. Navy is considering the development of electromagnetic railguns for future ships for naval surface-fire support (NSFS) and other missions. To reach long ranges, muzzle velocities in excess of 2000 m/s with projectile flight mass of 16 kg and above are needed. Relatively high firing rates are desired; typically 6-12 rounds per minute, so substantial power demands will be made on the ship. For an electrically driven ship, as in the DD(X) concepts presently being explored by the Navy, an existing electrical infrastructure would be in place that could be modified to recharge the pulsed power supplies for the railgun. This study focused on the railgun, the pulsed power system needed to drive the gun, the power system needed to restore the energy required to match the firing rate, and special ship interfaces. Railgun concepts and sizing for the NSFS mission were undertaken, and research and development issues of importance for the success of a future system were identified. Recommendations for analytical and experimental studies that should be undertaken to further address these issues are provided.

Heating at the root or throat position in C-shaped armatures is a concern because it can lead to cracking, melting, and premature failure. This paper presents results from experiments in which railgun armatures made of 7075 aluminum alloy were recovered after being launched at various levels of electrical action. Inspection of the recovered armatures showed reproducible patterns of damage at the root position that correlated with the severity of the launch conditions. Damage ranged from deformation and cracking at low levels of electrical action to melting to ejection of material and magnetic sawing at higher levels of action. There appears to be a threshold beyond which damage begins to propagate rapidly into the armature. The temperature rise in the armature was modeled with the three-dimensional (3-D) finite-element analysis (FEA) program EMAP3D. Our results show that it is possible to predict the onset of severe damage without modeling the complex phase-change behavior of the materials.

The majority of published work on the transition to arcing contact in solid-armature railguns has focused on modeling the current melt wave, a localized front of molten material that erodes the perimeter of the armature. The most frequently cited models are based on two-dimensional (2D) analytical formulations in which the dominant source of current concentration is due to the velocity skin effect (VSE). This paper compares the predictions of three such models with a 2D numerical simulation that includes the effects of VSE, melting, and loss of molten material.

This paper describes a continuation of earlier reported work in which C-armatures were launched and recovered for the purpose of studying melt-wave erosion (MWE) damage on the contact surface of the trailing arms. The purpose of the experiments reported here is to begin linking experimental results to theoretical predictions of the erosion process. Specifically, we sought to test a claim made in many of the two-dimensional (2-D) analytical models of MWE that the speed of the erosion front should be proportional to velocity and to the square of current. To this end, we performed two series of tests in which velocity and current were varied while controlling other variables. Peak currents ranged from 400 to 1200 kA, and exit speeds ranged from 100 to 700 m/s. Results reported include mass and volume of eroded material measured by mechanical profilometry and qualitative observations of the damage. The erosion rates are in reasonable agreement with the scaling relations predicted by 2-D theory. The erosion patterns, however, are qualitatively different from 2-D predictions.

This paper reviews fast iterative reweighted least squares (IRLS) and residual steepest descent (RSD) algorithms for L p , 1\leq p \leq2 , deconvolution. The timing aspects and the implementation of the IRLS algorithm on an array processor are discussed. The effectiveness of L 1 deconvolution and its insensitivity to noise bursts are illustrated using simgle synthetic as well as complex seismic data. Finally, it is shown that the L p prediction filters are stable as long as p \neq 1 .

The cellular prion protein (PrP(C)) is essential for the pathogenesis and transmission of prion diseases. Whereas the majority of PrP(C) is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor, a secreted form of the protein has been identified. Here we show that PrP(C) can be shed into the medium of human neuroblastoma SH-SY5Y cells by both protease- and phospholipase-mediated mechanisms. The constitutive shedding of PrP(C) was inhibited by a range of hydroxamate-based zinc metalloprotease inhibitors in a manner identical to the alpha-secretase-mediated shedding of the amyloid precursor protein, indicating a proteolytic shedding mechanism. Like amyloid precursor protein, this zinc metalloprotease-mediated shedding of PrP(C) could be stimulated by phorbol myristate acetate and by copper ions. The lipid raft-disrupting agents filipin and methyl-beta-cyclodextrin promoted the shedding of PrP(C) via a distinct mechanism that was not inhibited by hydroxamate-based inhibitors. Filipin-mediated shedding of PrP(C) is likely to occur via phospholipase cleavage of the GPI anchor, since a transmembrane polypeptide-anchored PrP construct was not shed in response to filipin treatment. Collectively, our data indicate that shedding of PrP(C) can occur via both secretase-like proteolytic cleavage of the protein and phospholipase cleavage of the GPI anchor moiety.

Activation of the cell-surface receptor Fas can lead to apoptosis in parenchymal cells in the liver, and if severe enough, result in fulminant hepatic failure and animal death. In the present study, we have examined the roles played by the Bcl-2 family members Bcl-xL and Bid in regulating this response. To do this, we have developed chemically modified 2'-O-(2-methoxy) ethyl antisense inhibitors of both Bid and Bcl-xL expression. In Balb/c mice, dosing with these antisense oligonucleotides reduced expression of the targeted mRNA by greater than 80% in the liver. This reduction was highly dependent upon oligonucleotide sequence and oligonucleotide dose. Reduction of Bcl-xL expression resulted in a potentiation of Fas-mediated apoptosis in liver and significant increase of the lethality of Fas-mediated fulminant hepatitis (p < 0.0001). In contrast, reduction of Bid expression protected the animals against Fas-mediated fulminant hepatitis and death (p < 0.0001). Simultaneous dosing of mice with Bcl-xL and Bid-targeting antisense oligonucleotides resulted in an inhibition of expression of both targeted proteins and protection of the animals from Fas-mediated apoptosis. These results demonstrate, for the first time, the role of Bcl-xL in regulating responses to proapoptotic Fas signaling in mouse liver. In addition, this is the first reported example demonstrating the ability of antisense inhibitors to reduce expression of multiple proteins in animals by simultaneous dosing.

The role of the N-terminal half of the prion protein (PrPC) in normal cellular function and pathology remains enigmatic. To investigate the biological role of the N-terminus of PrP, we examined the cellular properties of a construct of murine PrP, PrP-DA, in which the N-terminus is tethered to the membrane by an uncleaved signal peptide and which retains the glycosyl-phosphatidylinositol anchor. Human neuroblastoma SH-SY5Y cells expressing PrP-DA were more susceptible to hydrogen peroxide and copper induced toxicity than wtPrP expressing cells. The PrP-DA expressing cells had an increased level of intracellular free radicals and reduced levels of superoxide dismutase and glutathione peroxidase as compared to the wtPrP expressing cells. The membrane topology, cell surface location, lipid raft localisation, intracellular trafficking and copper-mediated endocytosis of PrP-DA were not significantly different from wtPrP. However, cells expressing PrP-DA accumulated an N-terminal fragment that was resistant to proteinase K. The data presented here are consistent with the N-terminal region of PrPC having a role in the cellular response to oxidative stress, and that tethering this region of the protein to the membrane compromises this function through the accumulation of a protease-resistant N-terminal fragment, similar to that seen in some forms of human prion disease.

Extracellular adenosine triphosphate (ATP), acting at P2X ionotropic receptors, is implicated in numerous sensory processes. Exogenous ATP has been shown to be algogenic in both animals and humans. Research focus has been directed towards the P2X(3) receptor, as it is preferentially expressed on nociceptive C-fibers and its implication in pain processing is supported by an altered nociceptive phenotype in P2X(3) knock-out mice. In order to further characterize the role of P2X(3) receptor activation in nociception, we evaluated the effects of continuous intrathecal administration of P2X(3) antisense oligonucleotides for 7 days in the rat. P2X(3) receptor antisense oligonucleotide treatment significantly decreased nociceptive behaviors observed after injection of complete Freund's adjuvant (CFA), formalin or alphabeta-methylene ATP into the rat's hind paw. The anti-hyperalgesic effects of the antisense treatment in the CFA model of inflammatory pain were dose related. Similar effects were observed with two distinct P2X(3) antisense oligonucleotides. These behavioral effects were significantly correlated with a decrease in P2X(3) receptor protein expression in the dorsal root ganglia (DRG). In contrast, a decrease in P2X(3) receptor protein expression in the DRG did not affect nociceptive behavior in the carrageenan model of acute thermal hyperalgesia. P2X(3) receptor antisense oligonucleotide treatment also significantly reduced mechanical allodynia observed after spinal nerve ligation. Overall, the present data demonstrate that activation of P2X(3) receptors contribute to the expression of chronic inflammatory and neuropathic pain states and that relief form these forms of chronic pain might be achieved by selective blockade of P2X(3 )receptor expression or activation.

The ability to deliver genes as therapeutics requires an understanding of the vector pharmacokinetics similar to that required for conventional drugs. A first question is the half-life of the vector in the bloodstream. Retroviral vectors produced in certain human cell lines differ from vectors produced in nonhuman cell lines in being substantially resistant to inactivation in vitro by human serum complement (F. L. Cosset, Y. Takeuchi, J. L. Battini, R. A. Weiss, and M. K. Collins, J. Virol. 69:7430–7436, 1995). Thus, use of human packaging cell lines (PCL) may produce vectors with longer half-lives, resulting in more-efficacious in vivo gene therapy. However, survival of human PCL-produced vectors in vivo following systemic administration has not been explored. In this investigation, the half-lives of retroviral vectors packaged by either canine D17 or human HT1080 PCL were measured in the bloodstreams of macaques and chimpanzees. Human PCL-produced vectors exhibited significantly higher concentrations of circulating biologically active vector at the earliest time points measured (>1,000-fold in chimpanzees), as well as substantially extended half-lives, compared to canine PCL-produced vectors. In addition, the circulation half-life of human PCL-produced vector was longer in chimpanzees than in macaques. This was consistent with in vitro findings which demonstrated that primate serum inactivation of vector produced from human PCL increased with increasing phylogenetic distance from humans. These results establish that in vivo retroviral vector half-life correlates with in vitro resistance to complement. Furthermore, these findings should influence the choice of animal models used to evaluate retroviral-vector-based therapies.

Multiple organ failure (MOF) is accompanied by muscle wasting, but changes in body composition are frequently obscured by fluid retention (edema), mainly in superficial and visceral tissue. There is a need to assess body composition and changes in body composition in these circumstances independently of edema. A relation was sought between fat-free (lean tissue) mass [calculated from body weight and skinfold thicknesses and measured by using dual-energy X-ray absorptiometry (DXA)] and muscle thickness (measured using ultrasound at a variety of sites accessible in an unconscious supine subject) to determine which sites correlated best with lean body mass. The three best sites were midbiceps, midforearm anteriorly, and midthigh anteriorly: R2 for the simple sum of the three sites correlated with fat-free mass from skinfold thicknesses was 71.1%, and with lean tissue mass from DXA was 76.1%. Serial measurements of both muscle thickness and midupper-arm circumference in nine patients with MOF showed a complete dissociation; in all nine there was a significant negative correlation of muscle thickness with time (P < 0.05) but changes in arm circumference were random. Only one patient showed a significant negative correlation with time, seven showed no change, and one other showed a significant increase. The muscle thicknesses that correlate best with lean body mass are measured over the biceps, anterior forearm, and anterior thigh. Monitoring muscle thicknesses at these three sites identifies wasting in edematous patients as it is happening.

Abstract 481 Hepcidin, the master regulator of iron homeostasis, is a peptide that is mainly expressed and secreted by the liver. Low levels of hepcidin are associated with increased iron absorption. In conditions in which hepcidin is chronically repressed, such as hereditary hemochromatosis and b-thalassemia, patients suffer from iron overload and very severe pathophysiological sequelae associated with this condition. Hepcidin expression is regulated predominantly at the transcriptional level by multiple factors. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor of hepcidin expression. It has been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of Tmprss6 expression in hereditary hemochromatosis (Hfe−/−) mice ameliorates the iron overload phenotype (Finberg et al. Nature Genetics, 2008; Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg et al., Blood, 2011). It has also been demonstrated that hepcidin up-regulation using either a hepcidin transgene or Tmprss6−/− significantly improves iron overload and anemia in a mouse model of β-thalassemia intermedia (th3/+ mice) (Gardenghi et al. JCI, 120:4466, 2010; Nai et al. Blood, 119: 5021, 2012). In this report, we have examined whether reduction of Tmprss6 expression using antisense technology is an effective approach for the treatment of hereditary hemochromatosis and β-thalassemia. Second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6 were identified. When normal male C57BL/6 mice were treated with 25, 50 and 100mg/kg/week ASO for four weeks, we achieved up to >90% reduction of liver Tmprss6 mRNA levels and up to 5-fold induction of hepcidin mRNA levels in a dose-dependent manner. Dose-dependent reductions of serum iron and transferrin saturation were also observed. ASOs were well tolerated in these animals. In Hfe−/− mice (both males and females), ASOs were administrated at 100 mg/kg for six weeks. This treatment normalized transferrin saturation (from 92% in control animals to 26% in treatment group) and significantly reduced serum iron (from >300ug/dl in control group to The mouse model of thalassemia intermedia that we utilized mimics a condition defined as non-transfusion dependent thalassemia (NTDT) in humans. These patients exhibit increased iron absorption and iron overload due to ineffective erythropoiesis and suppression of hepcidin; iron overload is the most frequent cause of morbidity and mortality. Th3/+ animals exhibit ineffective erythropoiesis, characterized by increased proliferation and decreased differentiation of the erythroid progenitors, apoptosis of erythroblasts due to the presence of toxic hemichromes, reticulocytosis and shorter lifespan of red cells in circulation, leading to splenomegaly, extramedullary hematopoiesis and anemia (∼ 8 g/dL; Libani et al, Blood 112(3):875–85, 2008). Five month old th3/+ mice (both males and females) were treated with Tmprss6 ASO for six weeks. In th3/+ mice, ∼85% Tmprss6 reduction led to dramatic reductions of serum transferrin saturation (from 55–63% in control group down to 20–26% in treatment group). Liver iron concentration (LIC) was also greatly reduced (40–50%). Moreover, anemia endpoints were significantly improved with ASO treatment, including increases in red blood cells (∼30–40%), hemoglobin (∼2 g/dl), and hematocrit (∼20%); reduction of splenomegaly (∼50%); decrease of serum erythropoietin levels (∼50%); improved erythroid maturation as indicated by a strong reduction in reticulocyte number (50–70%) and in a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. Hemichrome analysis showed a significant decrease in the formation of toxic alpha-globin/heme aggregates associated with the red cell membrane. This was consistent with a remarkable improvement of the red cell distribution width (RDW) as well as morphology of the erythrocytes. In conclusion, these data demonstrate that targeting TMPRSS6 using antisense technology is a promising novel therapy for the treatment of hereditary hemochromatosis and β-thalassemia. Disclosures: Guo: Isis Pharmaceuticals: Employment. Booten:Isis Pharmaceuticals: Employment. Watt:Isis Pharmaceuticals: Employment. Freier:Isis Pharmaceuticals: Employment. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding. Monia:Isis Pharmaceuticals: Employment.

Zinc is released into the synaptic cleft upon exocytotic stimuli, although the mechanism for its reuptake into neurons is unresolved. Here we show that the cellular prion protein enhances the uptake of zinc into neuronal cells. This prion-protein-mediated zinc influx requires the octapeptide repeats and amino-terminal polybasic region in the prion protein, but not its endocytosis. Selective antagonists of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors block the prion protein-mediated zinc uptake, and the prion protein co-immunoprecipitates with both GluA1 and GluA2 AMPA receptor subunits. Zinc-sensitive intracellular tyrosine phosphatase activity is decreased in cells expressing prion protein and increased in the brains of prion-protein-null mice, providing evidence of a physiological consequence of this process. Prion protein-mediated zinc uptake is ablated in cells expressing familial associated mutants of the protein and in prion-infected cells. These data suggest that alterations in the cellular prion protein-mediated zinc uptake may contribute to neurodegeneration in prion and other neurodegenerative diseases., Prion proteins are implicated in a range of neurodegenerative diseases, which are, in part, due to a disruption of metal homeostasis. Watt et al. use selective antagonists to show that prion proteins mediate zinc uptake by interacting with GluA2-lacking, GluA1-containing AMPA receptors.

The unpredictable past and future of the prescription benefit business has resulted in an acute interest by plan sponsors in the concept of capitation. Capitation is effectively an insurance policy, offered by the provider, which will guarantee the cost of the prescription benefit. Such guarantees relative to the cost of the plan are attractive to the sponsor because they minimize risk and facilitate rate setting for HMOs and PPOs--and facilitate budgeting for corporate sponsors. Is capitation a cost-effective win-win alternative to traditional average wholesale price-based arrangements?

Model biomaterial surfaces with well defined chemistry were prepared from close-packed alkyltrichlorosilane monolayers on polished silicon and glass. The outermost molecular groups which come in direct contact with the biological environment were varied across a wide range of oxidation states by employing CF3, CH3, CO2CH3, and CH2OH terminal functionalities. Characterization by contact angles, surface spectroscopy, and ellipsometry verified that these model surfaces could be repeatedly prepared with good consistency for routine use to study biomolecule adsorption onto model surfaces. Adhesion of canine endothelial cells and the adsorption of proteins (human serum albumin and human fibrinogen) as well as a series of synthetic defined oligopeptides to these model surfaces have been studied. Endothelial cells attachment and growth were in the rank order of: CH2OH > CO2Me > CH3 > CF3. The peptides were comprised of different alternating sequences of lysine, leucine, and tryptophan residues. These structural differences imparted different amphiphilic characters that led to measurable differences in the adsorption of these peptides to liquid-vapor interfaces. The adsorption to model surfaces was studied using ESCA, radiometry, and concentration-dependent contact angles. ESCA and radiometry measured irreversible biomolecules adsorption whereas the contact angle method measured steadystate adsorption. Radiometric results were inconsistent with ESCA, possibly due to artifacts associated with protein radiolabeling. © 1993 John Wiley & Sons, Inc.

FXI, a component of the intrinsic pathway of coagulation, contributes to the generation of thrombin and is also activated by thrombin on the surface of platelets, resulting in further amplification of the coagulation process. Reports have shown that FXI deficiency in mice or in humans is generally not associated with major bleeding yet is associated with reduced risk for thromboembolic disease. To determine whether targeting FXI with antisense oligonucleotides (ASOs) can produce antithrombotic activity without increasing risk of bleeding, a series of 2nd Generation 2′-O-methoxyethyl modified ASOs were identified that specifically reduce levels of FXI in mice and were evaluated for antithrombotic activity and bleeding potential. Mice were dosed with FXI ASO (ISIS 404071) subcutaneously for three weeks. FXI ASO treatment resulted in a dosedependent and specific reduction in FXI mRNA levels in liver and FXI protein levels in plasma with an ED50 of 3.2 mg/kg. These reductions in FXI levels correlated well with a prolongation of aPTT with no effects on PT prolongation. The anticoagulant effect of FXI ASO treatment also correlated well with antithrombotic activity in a 10% FeCl3 induced IVC thrombosis mouse model across a wide dose range. Furthermore, FXI ASO treatment was generally well tolerated and no prolongation of tail bleeding time was observed at any dose tested, indicating a broad safety margin for FXI ASO targeting. Additional studies using intravital microscopy to study the effects of FXI ASO treatment on thrombosis formation in the mesenteric vein following 10% FeCl3 injury demonstrated that FXI ASO treatment significantly impeded platelet aggregation at the site of vessel injury, thrombus growth and vessel occlusion. Studies to further evaluate the safety of FXI targeting were conducted using a mouse model of intracerebral hemorrhage induced by intracranial injection of collagenase. In this model, FXI ASO treatment had no effect on hemorrhagic size, neurologic deficit score, or mortality at doses well above that needed to produce maximal antithrombotic activity. These results strongly suggest that FXI is an effective and safe target for antithrombotic therapy and that antisense drug technology could be an attractive approach to specifically target FXI for the treatment of thrombotic disorders.

A three-dimensional (3D) geologic model of part of the northern Nevada rift encompassing the Beowawe geothermal system was developed from a series of two-dimensional (2D) geologic and geophysical models. The 3D model was constrained by local geophysical, geologic, and drill-hole information and integrates geologic and tectonic interpretations for the region. It places important geologic constraints on the extent and configuration of the active Beowawe geothermal system. The geologic framework represented in this model facilitates hydrologic modeling of the Beowawe geothermal system and evaluation of fluid flow in faults and adjacent rock units. Basin depths were determined using an iterative gravity-inversion technique that calculates the thickness of low-density, basin-filling deposits. The remaining subsurface structure was modeled using 2D potential-field modeling software. Crustal cross sections from the 2D models were generalized for use in the 3D model and consist of six stratigraphic layers defined as low-density basin sediments, volcanic rocks, basalt-andesite rocks of the northern Nevada rift, Jurassic and Cretaceous intrusive rocks, and Paleozoic siliceous and carbonate sedimentary rocks of the upper and lower plates of the Roberts Mountains allochthon, respectively. This simplified stratigraphy was combined with mapped surface geology and was extrapolated across the 3D model area. Features along the northern Nevada rift depicted by the model may represent preexisting crustal structures that controlled the locations and character of Tertiary tectonic and magmatic events related to Basin and Range extension and emplacement of the middle Miocene northern Nevada rift. Several of the geologic features represented are important components of the Beowawe geothermal system. Prominent ENE-trending faults (e.g., Malpais fault) that bound the southern edge of Whirlwind Valley, and older NNW-striking faults (e.g., Dunphy Pass and Muleshoe faults) that form major features of the model, are likely important pathways for geothermal fluids and groundwater flow from the Humboldt River, which may recharge the Beowawe system.

Anemia is one of the more common blood disorders and is associated with a number of diseases, including chronic kidney disease, chronic inflammation, and certain types of cancer. Under these conditions iron is essential as it is required for erythroid progenitor cell proliferation and red cell function. Hepcidin is a liver-derived growth factor that regulates iron absorption in the GI tract and iron absorption and release in tissues. Furthermore, hepcidin overexpression has been strongly linked mechanistically as a mediator of decreased iron availability and anemia. We have utilized an antisense approach to investigate the role of hepcidin in animal models of anemia and as a potential therapeutic approach for the treatment of this disorder in humans. Second-generation 2′-O-methoxyethyl chimeric antisense oligonucleotides (ASOs) were screened in isolated primary mouse hepatocytes, followed by in vivo screening in mice, for the ability to reduce hepcidin mRNA levels. ASO treatment resulted in a reduction of hepcidin mRNA in liver which was associated with a significant increase in serum iron levels. The best hepcidin ASO was then tested in a mouse model of turpentine induced hypoferremia and anemia to determine the role of hepcidin in regulating serum iron and anemia endpoints. Mice were treated (I.P. twice/weekly) with hepcidin or control ASO for two weeks at varying doses prior to a single subcutaneous injection of turpentine. Turpentine treatment 16 hours post-injection produced a significant reduction in serum iron levels and at two weeks resulted in reduced RBC numbers, hematocrit and hemoglobin levels. Treatment with the hepcidin ASO resulted in a dose dependent improvement in all of these endpoints while the control oligonucleotide had no effect. Studies are in progress to further characterize the pharmacological activity of hepcidin ASO in additional models of anemia and results from these on-going studies will also be presented.

The PrP(C) [cellular isoform of PrP (prion protein)] can undergo a conformational conversion to produce a proteinase-resistant form PrP(Sc) (scrapie isoform of PrP), a step critical for the development of prion disease. Although essential for disease progression, the normal cellular function of PrP(C) remains unknown. Suggestions to date have centred on a protective role against oxidative stress. We have demonstrated that ROS (reactive oxygen species)-mediated beta-cleavage of PrP(C) occurs at the cell surface, can be inhibited following hydroxyl radical quenching and has a prerequisite for the octarepeat region in the N-terminus of the protein. Significantly, two disease-associated mutants of PrP, namely PG14 and A116V (Ala(116)-->Val), were unable to undergo beta-cleavage and this lack of proteolysis was accompanied by functional consequences in cells expressing these mutant proteins. The cells were found to be less viable following exposure to copper and H2O2, had reduced levels of glutathione peroxidase and increased amounts of intracellular oxygen radicals. These results suggest that beta-cleavage of PrP(C) is an initial consequence following exposure to ROS in the extracellular environment contributing to a pathway involved in antioxidant protection of neuronal cells.