Nr2e3 is a genetic modifier that rescues retinal degeneration and promotes homeostasis in multiple models of retinitis pigmentosa.

Recent advances in viral vector engineering, as well as an increased understanding of the cellular and molecular mechanism of retinal diseases, have led to the development of novel gene therapy approaches. Furthermore, ease of accessibility and ocular immune privilege makes the retina an ideal target for gene therapies. In this study, the nuclear hormone receptor gene Nr2e3 was evaluated for efficacy as broad-spectrum therapy to attenuate early to intermediate stages of retinal degeneration in five unique mouse models of retinitis pigmentosa (RP). RP is a group of heterogenic inherited retinal diseases associated with over 150 gene mutations, affecting over 1.5 million individuals worldwide. RP varies in age of onset, severity, and rate of progression. In addition, ~40% of RP patients cannot be genetically diagnosed, confounding the ability to develop personalized RP therapies. Remarkably, Nr2e3 administered therapy resulted in reduced retinal degeneration as observed by increase in photoreceptor cells, improved electroretinogram, and a dramatic molecular reset of key transcription factors and associated gene networks. These therapeutic effects improved retinal homeostasis in diseased tissue. Results of this study provide evidence that Nr2e3 can serve as a broad-spectrum therapy to treat multiple forms of RP.

Automation of the anthrone assay for carbohydrate concentration determinations.

Reported is the adaptation of a manual polysaccharide assay applicable for glycoconjugate vaccines such as Prevenar to an automated liquid handling system (LHS) for improved performance. The anthrone assay is used for carbohydrate concentration determinations and was scaled to the microtiter plate format with appropriate mixing, dispensing, and measuring operations. Adaptation and development of the LHS platform was performed with both dextran polysaccharides of various sizes and pneumococcal serotype 6A polysaccharide (PnPs 6A). A standard plate configuration was programmed such that the LHS diluted both calibration standards and a test sample multiple times with six replicate preparations per dilution. This extent of replication minimized the effect of any single deviation or delivery error that might have occurred. Analysis of the dextran polymers ranging in size from 214 kDa to 3.755 MDa showed that regardless of polymer chain length the hydrolysis was complete, as evident by uniform concentration measurements. No plate positional absorbance bias was observed; of 12 plates analyzed to examine positional bias the largest deviation observed was 0.02% percent relative standard deviation (%RSD). The high purity dextran also afforded the opportunity to assess LHS accuracy; nine replicate analyses of dextran yielded a mean accuracy of 101% recovery. As for precision, a total of 22 unique analyses were performed on a single lot of PnPs 6A, and the resulting variability was 2.5% RSD. This work demonstrated the capability of a LHS to perform the anthrone assay consistently and a reduced assay cycle time for greater laboratory capacity.

Identification of particulates in vaccine formulations containing aluminum phosphate.

Density gradient centrifugation was used for identification of particulates in vaccines formulated with aluminum phosphate (AlPO(4)) adjuvant. Protein-derived particulates are sporadically seen in protein pre-filled formulations where siliconized syringes are employed. These particulates are difficult to distinguish from the AlPO(4) suspension. Prior to centrifugation, Histopaque solution is placed at the bottom of the test tube (or the original syringe), resulting in a layer beneath the suspension. After brief centrifugation, AlPO(4) settles to the bottom of the test tube, while the light-weighted particulates in the sample become visible, floating in the inter-phase between the two layers.

Monitoring activation sites on polysaccharides by GC-MS.

A method has been developed to determine the location and order of activation for potential saccharide antigens used in conjugate vaccine development. Saccharides were monitored for activation by sodium periodate oxidation and subsequent analysis by gas chromatography-mass spectrometry (GC-MS). Pneumococcal serotype polysaccharides 7F and 18C were evaluated as polysaccharides containing multiple potential sites for activation. Sialyllactose was used as a model oligosaccharide compound to evaluate oxidation of terminally linked sialic acids and reducing sugar residues. Oxidized saccharides were analyzed by monosaccharide composition and/or linkage analysis to elucidate specific activation of cis versus trans diols, as well as diols containing primary versus secondary alcohols, at specified levels of periodate. Samples (100-500 microg) were sequentially oxidized, reduced, methanolyzed, and derivatized in a single reaction vial for routine analysis by GC-MS.

Addition of glycerol for improved methylation linkage analysis of polysaccharides.

A presolubilization procedure with the use of glycerol is shown to be applicable for the structural analysis of polysaccharides. Neutral, acidic, high-molecular-weight and low-molecular-weight polysaccharides were solubilized in glycerol prior to methylation and subsequent linkage analysis by GC-MS. All four types of polysaccharides showed significant increases in derivatization following presolubilization as measured by recovery of partially methylated alditol acetates.

Determination of saccharide content in pneumococcal polysaccharides and conjugate vaccines by GC-MSD.

A simple and sensitive gas chromatographic method was designed for quantitative analysis of Streptococcus pneumoniae capsular polysaccharides, activated polysaccharides, and polysaccharide conjugates. Pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F polysaccharide or conjugate were subjected to methanolysis in 3N hydrochloric acid in methanol followed by re-N-acetylation and trimethylsilylation. Derivatized samples were chromatographed and detected using gas chromatography with mass selective detector. Gas chromatographic results were compared with colorimetric values with agreement of 92 to 123% over the range of all samples tested. Monosaccharides released during methanolysis included hexoses, uronic acids, 6-deoxy-hexoses, amino sugars, and alditols. Quantitative recovery of monosaccharides was achieved for all serotypes by the use of a single methanolysis, derivatization, and chromatography procedure. Response factors generated from authentic monosaccharide standards were used for quantitation of pneumococcal polysaccharides and conjugates with confirmation of peak assignments by retention time and mass spectral analysis. This method allows saccharide quantitation in multivalent pneumococcal vaccine intermediates and final drug products with low-level detection (10 pg) and peak purity.