Identification of small interfering RNA targeting Signal Transducer and Activator of Transcription 6: Characterisation and selection of candidates for pre-clinical development

The interleukin (IL)-13 pathway and its associated transcription factor, signal transducer and activator of transcription 6 (STAT6), have been clearly implicated in the pathogenesis of bronchial asthma. We have developed a system to effectively screen the STAT6 gene for targeting with small interfering (si) RNA molecules. By incorporating an in silico and in vitro screening system we were able to identify fourteen siRNA molecules suitable for pre-clinical drug development. Furthermore, we were able to demonstrate that modification of certain siRNAs, designed to improve in vivo longevity, was possible without significant loss of target knockdown efficacy and that the siRNA produced by our selection process did not induce demonstrable interferon responses. These data suggest that several STAT6-targeting siRNA suitable for pre-clinical development are available for potential use in the treatment of asthma

Mice Engrafted with Human Fetal Thymic Tissue and Hematopoietic Stem Cells Develop Pathology Resembling Chronic Graft-versus-Host Disease

AbstractChronic graft-versus-host disease (cGVHD) is a significant roadblock to long-term hematopoietic stem cell (HSC) transplantation success. Effective treatments for cGVHD have been difficult to develop, in part because of a paucity of animal models that recapitulate the multiorgan pathologies observed in clinical cGVHD. Here we present an analysis of the pathology that occurs in immunodeficient mice engrafted with human fetal HSCs and implanted with fragments of human fetal thymus and liver. Starting at time points generally later than 100 days post-transplantation, the mice developed signs of illness, including multiorgan cellular infiltrates containing human T cells, B cells, and macrophages; fibrosis in sites such as lungs and liver; and thickened skin with alopecia. Experimental manipulations that delayed or reduced the efficiency of the HSC engraftment did not affect the timing or progression of disease manifestations, suggesting that pathology in this model is driven more by factors associated with the engrafted human thymic organoid. Disease progression was typically accompanied by extensive fibrosis and degradation of the thymic organoid, and there was an inverse correlation of disease severity with the frequency of FoxP3+ thymocytes. Hence, the human thymic tissue may contribute T cells with pathogenic potential, but the generation of regulatory T cells in the thymic organoid may help to control these cells before pathology resembling cGVHD eventually develops. This model thus provides a new system to investigate disease pathophysiology relating to human thymic events and to evaluate treatment strategies to combat multiorgan fibrotic pathology produced by human immune cells

The effects of frequent nocturnal home hemodialysis: the Frequent Hemodialysis Network Nocturnal Trial

Prior small studies have shown multiple benefits of frequent nocturnal hemodialysis compared to conventional three times per week treatments. To study this further, we randomized 87 patients to three times per week conventional hemodialysis or to nocturnal hemodialysis six times per week, all with single-use high-flux dialyzers. The 45 patients in the frequent nocturnal arm had a 1.82-fold higher mean weekly stdKt/Vurea, a 1.74-fold higher average number of treatments per week, and a 2.45-fold higher average weekly treatment time than the 42 patients in the conventional arm. We did not find a significant effect of nocturnal hemodialysis for either of the two coprimary outcomes (death or left ventricular mass (measured by MRI) with a hazard ratio of 0.68, or of death or RAND Physical Health Composite with a hazard ratio of 0.91). Possible explanations for the left ventricular mass result include limited sample size and patient characteristics. Secondary outcomes included cognitive performance, self-reported depression, laboratory markers of nutrition, mineral metabolism and anemia, blood pressure and rates of hospitalization, and vascular access interventions. Patients in the nocturnal arm had improved control of hyperphosphatemia and hypertension, but no significant benefit among the other main secondary outcomes. There was a trend for increased vascular access events in the nocturnal arm. Thus, we were unable to demonstrate a definitive benefit of more frequent nocturnal hemodialysis for either coprimary outcome

Analysis of the CD1 Antigen Presenting System in Humanized SCID Mice

CD1 molecules are glycoproteins that present lipids and glycolipids for recognition by T cells. CD1-dependent immune activation has been implicated in a wide range of immune responses, however, our understanding of the role of this pathway in human disease remains limited because of species differences between humans and other mammals: whereas humans express five different CD1 gene products (CD1a, CD1b, CD1c, CD1d, and CD1e), muroid rodents express only one CD1 isoform (CD1d). Here we report that immune deficient mice engrafted with human fetal thymus, liver, and CD34+ hematopoietic stem cells develop a functional human CD1 compartment. CD1a, b, c, and d isoforms were highly expressed by human thymocytes, and CD1a+ cells with a dendritic morphology were present in the thymic medulla. CD1+ cells were also detected in spleen, liver, and lungs. APCs from spleen and liver were capable of presenting bacterial glycolipids to human CD1-restricted T cells. ELISpot analyses of splenocytes demonstrated the presence of CD1-reactive IFN-γ producing cells. CD1d tetramer staining directly identified human iNKT cells in spleen and liver samples from engrafted mice, and injection of the glycolipid antigen α-GalCer resulted in rapid elevation of human IFN-γ and IL-4 levels in the blood indicating that the human iNKT cells are biologically active in vivo. Together, these results demonstrate that the human CD1 system is present and functionally competent in this humanized mouse model. Thus, this system provides a new opportunity to study the role of CD1-related immune activation in infections to human-specific pathogens

Development of Pre-Clinical Models for Evaluating the Therapeutic Potential of Candidate siRNA Targeting STAT6

As the development of siRNA therapeutics is technically challenging, this study evaluates various pre-clinical models in their ability to suitably test candidate siRNA for respiratory administration. This study shows that use of chemically-modified siRNA was not associated with cellular toxicity, yet enhanced bio-availability without evidence of reduced efficacy. Furthermore, we established a robust, sensitive method for determining siRNA bio-distribution in vivo, and co-developed a novel nasal administration model to further aid evaluation

Nucleic Acid Polymers with Accelerated Plasma and Tissue Clearance for Chronic Hepatitis B Therapy

REP 2139 is a nucleic acid polymer (NAP) currently under clinical development for chronic hepatitis B (HBV) therapy. This preclinical study investigated different REP 2139 analogs that would display reduced accumulation in the serum and tissues, while retaining an antiviral effect against HBV infection. REP 2139 analogs were evaluated in human plasma, CD-1 mice, cynomolgus monkeys, and Pekin ducks. Discrete ribose transformation to 2′OH in selected riboadenosines resulted in a slow degradation in acidified human plasma that plateaued after 48 hr. REP 2165, a REP 2139 analog containing three unmodified riboadenosines equally spaced throughout the polymer, showed similar plasma clearance and tissue distribution as REP 2139 in mice and cynomolgus monkeys after a single dose. Interestingly, after repeated administration, accumulation of REP 2165 in plasma and organs was reduced, indicating a dramatically faster rate of clearance from organs after therapy was ended in both species. Both REP 2139 and REP 2165 were well tolerated at clinically relevant doses, with no alterations in liver, kidney, or hematological function. In chronic duck HBV (DHBV) infection, REP 2165 displayed significantly reduced liver accumulation after repeated dosing but retained antiviral activity similar to REP 2139. These results indicate the therapeutic potential of REP 2165 against chronic HBV infection in patients is similar to REP 2139, but with significantly reduced drug accumulation and improved tissue clearance. Keywords: nucleic acid polymer, HBV, pharmacokinetic

Expression of CD1c enhances human invariant NKT cell activation by α-GalCer

Invariant natural killer T (iNKT) cells are innate T lymphocytes that specifically recognize α-linked glycosphingolipids (α-GSLs) as antigens presented by CD1d molecules. Activating iNKT cells by administering α-GSLs improves disease outcomes in murine cancer models and, thus, there is great interest in the clinical potential of these lipids for treating human cancers. However, humans possess several other CD1 isoforms that are not present in mice and it is not clear whether these CD1 molecules, which also bind lipids, affect human iNKT cell responses. We demonstrate here that CD1c, which is co-expressed with CD1d on blood dendritic cells and on a fraction of B cells, is able to present α-galactosylceramide (α-GalCer) as a weak agonist to human iNKT cells, and that the presence of CD1c synergistically enhances α-GalCerdependent activation of iNKT cells by CD1d. Primary human B cells expressing CD1c induced stronger iNKT cell responses to α-GalCer than the CD1c- subset, and an antibody against CD1c inhibited iNKT cell cytokine secretion. These results suggest that therapeutic activation of human iNKT cells by α-GSLs will be driven preferentially by CD1c+ cell types. Thus, B cell neoplasias that co-express CD1c and CD1d may be particularly susceptible to α-GSL therapy, and cancer vaccines using α-GSLs as adjuvants may be most effective when presented by CD1c+ antigen-presenting cells

Effectiveness of STAT6 targeting siRNA within a rat model of allergic inflammation.

<p>Ovalbumin-sensitised animals were administered 372u, 372m, MMC siRNA (2 mg/kg, intra-tracheal) or dexamethasone (0.3 mg/kg) on 3 consecutive days and then aerosol challenged with ovalbumin 24 hours after the final dose. Seventy-two hours after challenge, 372m was present at significantly higher concentrations in BAL than 372u (a, b). Treatment with 372u or 372m did not reduce lung inflammation compared to the saline treated control, as evidenced by histological scoring (c) and inflammatory cell enumeration (d-f). Treatment with dexamethasone however, did significantly reduce lung inflammation following allergen challenge. Values presented are mean ± S.E.M (n = 10), and P-value represents comparison to the saline treated control.</p