Sea Lane Biotechnologies (United States)

Three critical advances in simplifying the adoption of P(V)-based stereopure, phosphorothioate-containing oligonu-cleotide synthesis are reported. A more inexpensive phosphorus-sulfur incorporation reagent (Ψ-Br) is introduced, a robust linker system was developed, and a systematic study of common nucleobase protecting groups performed to significantly reduce the barrier to adoption of this technology.

To develop a method for modulation of transgene expression in retinal pigment epithelium (RPE) using scanning laser that spares neurosensory retina. Fifteen pigmented rabbits received subretinal injection of recombinant adeno-associated virus (rAAV-2) encoding green fluorescent protein (GFP). GFP expression was measured using confocal scanning laser ophthalmoscopy (cSLO) fluorescence imaging and immunohistochemistry. To reduce the total expression in RPE by half, 50% of the transfected RPE cells were selectively destroyed by microsecond exposures to scanning laser with 50% pattern density. The selectivity of RPE destruction and its migration and proliferation were monitored using fluorescein angiography, spectral-domain optical coherence tomography (SD-OCT), and light, transmission, and scanning electron microscopy. 5-Bromo-2'-dioxyuridine (BrdU) assay was performed to evaluate proliferation of RPE cells. RPE cells were selectively destroyed by the line scanning laser with 15 μs exposures, without damage to the photoreceptors or Bruch's membrane. RPE cells started migrating after the first day, and in 1 week there was complete restoration of RPE monolayer. Selective laser treatment decreased the GFP fluorescence by 54% as compared to control areas; this was further decreased by an additional 48% following a second treatment 1 month later. BrdU assay demonstrated proliferation in approximately half of the RPE cells in treatment areas. Microsecond exposures produced by scanning laser destroyed RPE cells selectively, without damage to neural retina. Continuity of RPE layer is restored within days by migration and proliferation, but transgene not integrated into the nucleus is not replicated. Therefore, gene expression can be modulated in a precise manner by controlling the laser pattern density and further adjusted using repeated applications.

Improper regulation of B cell responses leads to excessive production of antibodies and contributes to the development of autoimmune disease. T helper 17 (Th17) cells also drive the development of autoimmune disease, but the role of B cells in shaping Th17 cell-mediated immune responses, as well as the reciprocal regulation of B cell responses by IL-17 family cytokines, remains unclear. The aim of this study was to characterize the regulation of IL-17A and IL-17F in a model of T cell-dependent B cell activation. Stimulation of primary human B cell and peripheral blood mononuclear cell (BT) co-cultures with α-IgM and a non-mitogenic concentration of superantigens for three days promoted a Th17 cell response as evidenced by increased expression of Th17-related gene transcripts, including Il17f, Il21, Il22, and Il23r, in CD4 T cells, as well as the secretion of IL-17A and IL-17F protein. We tested the ability of 144 pharmacologic modulators representing 91 different targets or pathways to regulate IL-17A and IL-17F production in these stimulated BT co-cultures. IL-17A production was found to be preferentially sensitive to inhibition of the PI3K/mTOR pathway, while prostaglandin EP receptor agonists, including PGE2, increased IL-17A concentrations. In contrast, the production of IL-17F was inhibited by PGE2, but selectively increased by TLR2 and TLR5 agonists. These results indicate that IL-17A regulation is distinct from IL-17F in stimulated BT co-cultures and that this co-culture approach can be used to identify pathway mechanisms and novel agents that selectively inhibit production of IL-17A or IL-17F.

Influenza viruses elude immune responses and antiviral chemotherapeutics through genetic drift and reassortment. As a result, the development of new strategies that attack a highly conserved viral function to prevent and/or treat influenza infection is being pursued. Such novel broadly acting antiviral therapies would be less susceptible to virus escape and provide a long lasting solution to the evolving virus challenge. Here we report the in vitro and in vivo activity of a human monoclonal antibody (A06) against two isolates of the 2009 H1N1 pandemic influenza virus. This antibody, which was obtained from a combinatorial library derived from a survivor of highly pathogenic H5N1 infection, neutralizes H5N1, seasonal H1N1 and 2009 “Swine” H1N1 pandemic influenza in vitro with similar potency and is capable of preventing and treating 2009 H1N1 influenza infection in murine models of disease. These results demonstrate broad activity of the A06 antibody and its utility as an anti-influenza treatment option, even against newly evolved influenza strains to which there is limited immunity in the general population.