Preclinical evaluation of ADVM-062, a novel intravitreal gene therapy vector for the treatment of blue cone monochromacy.

Blue cone monochromacy (BCM) is a rare X-linked retinal disease characterized by the absence of L- and M-opsin in cone photoreceptors, considered a potential gene therapy candidate. However, most experimental ocular gene therapies utilize subretinal vector injection which would pose a risk to the fragile central retinal structure of BCM patients. Here we describe the use of ADVM-062, a vector optimized for cone-specific expression of human L-opsin and administered using a single intravitreal (IVT) injection. Pharmacological activity of ADVM-062 was established in gerbils, whose cone-rich retina naturally lacks L-opsin. A single IVT administration dose of ADVM-062 effectively transduced gerbil cone photoreceptors and produced a de novo response to long-wavelength stimuli. To identify potential first-in-human doses we evaluated ADVM-062 in non-human primates. Cone-specific expression of ADVM-062 in primates was confirmed using ADVM-062.myc, a vector engineered with the same regulatory elements as ADVM-062. Enumeration of human OPN1LW.myc-positive cones demonstrated that doses ≥3 × 1010 vg/eye resulted in transduction of 18%-85% of foveal cones. A Good Laboratory Practice (GLP) toxicology study established that IVT administration of ADVM-062 was well tolerated at doses that could potentially achieve clinically meaningful effect, thus supporting the potential of ADVM-062 as a one-time IVT gene therapy for BCM.

Using habitat suitability models to target invasive plant species surveys.

Managers need new tools for detecting the movement and spread of nonnative, invasive species. Habitat suitability models are a popular tool for mapping the potential distribution of current invaders, but the ability of these models to prioritize monitoring efforts has not been tested in the field. We tested the utility of an iterative sampling design (i.e., models based on field observations used to guide subsequent field data collection to improve the model), hypothesizing that model performance would increase when new data were gathered from targeted sampling using criteria based on the initial model results. We also tested the ability of habitat suitability models to predict the spread of invasive species, hypothesizing that models would accurately predict occurrences in the field, and that the use of targeted sampling would detect more species with less sampling effort than a nontargeted approach. We tested these hypotheses on two species at the state scale (Centaurea stoebe and Pastinaca sativa) in Wisconsin (USA), and one genus at the regional scale (Tamarix) in the western United States. These initial data were merged with environmental data at 30-m2 resolution for Wisconsin and 1-km2 resolution for the western United States to produce our first iteration models. We stratified these initial models to target field sampling and compared our models and success at detecting our species of interest to other surveys being conducted during the same field season (i.e., nontargeted sampling). Although more data did not always improve our models based on correct classification rate (CCR), sensitivity, specificity, kappa, or area under the curve (AUC), our models generated from targeted sampling data always performed better than models generated from nontargeted data. For Wisconsin species, the model described actual locations in the field fairly well (kappa = 0.51, 0.19, P < 0.01), and targeted sampling did detect more species than nontargeted sampling with less sampling effort (chi2 = 47.42, P < 0.01). From these findings, we conclude that habitat suitability models can be highly useful tools for guiding invasive species monitoring, and we support the use of an iterative sampling design for guiding such efforts.

Leaf Morphological and Nutrient Traits of Common Woody Plants Change Along the Urban-Rural Gradient in Beijing, China.

An increasing number of studies have found differences in the diversity of plant functional traits between urban and rural sites as a result of urbanization. However, the results remain inconsistent. In this study, we measured morphological and nutrient traits of 11 common woody plants along a continuous urban-rural gradient in Beijing, China. Leaf size (e.g., length, width, and area), specific leaf area, and leaf nitrogen and potassium contents decreased gradually and significantly along the urban-rural gradient, indicating that urbanization can enhance the capacity of plants to acquire resources for growth and production. Furthermore, soil nutrients and air temperature decreased along the urban-rural gradient, while air relative humidity increased. A structural equation model showed that these alterations in physical factors attributable to urbanization contributed directly or indirectly to changes in leaf functional traits, implying that changes in soil nutrients and micro-climate induced by urbanization may affect plant growth and production because of the improvement in resource acquisition capacity.

2A4 binds soluble and insoluble light chain aggregates from AL amyloidosis patients and promotes clearance of amyloid deposits by phagocytosis †.

Amyloid light chain (AL) amyloidosis is characterized by misfolded light chain (LC) (amyloid) deposition in various peripheral organs, leading to progressive dysfunction and death. There are no regulatory agency-approved treatments for AL amyloidosis, and none of the available standard of care approaches directly targets the LC protein that constitutes the amyloid. NEOD001, currently in late-stage clinical trials, is a conformation-specific, anti-LC antibody designed to specifically target misfolded LC aggregates and promote phagocytic clearance of AL amyloid deposits. The present study demonstrated that the monoclonal antibody 2A4, the murine form of NEOD001, binds to patient-derived soluble and insoluble LC aggregates and induces phagocytic clearance of AL amyloid in vitro. 2A4 specifically labeled all 21 fresh-frozen organ samples studied, which were derived from 10 patients representing both κ and λ LC amyloidosis subtypes. 2A4 immunoreactivity largely overlapped with thioflavin T-positive labeling, and 2A4 bound both soluble and insoluble LC aggregates extracted from patient tissue. Finally, 2A4 induced macrophage engagement and phagocytic clearance of AL amyloid deposits in vitro. These findings provide further evidence that 2A4/NEOD001 can effectively clear and remove human AL-amyloid from tissue and further support the rationale for the evaluation of NEOD001 in patients with AL amyloidosis.

Transgenic mice expressing human fibroblast growth factor-19 display increased metabolic rate and decreased adiposity.

The fibroblast growth factors (FGFs), and the corresponding receptors, are implicated in more than just the regulation of epithelial cell proliferation and differentiation. Specifically, FGF23 is a regulator of serum inorganic phosphate levels, and mice deficient in FGF receptor-4 have altered cholesterol metabolism. The recently described FGF19 is unusual in that it is nonmitogenic and appears to interact only with FGF receptor-4. Here, we report that FGF19 transgenic mice had a significant and specific reduction in fat mass that resulted from an increase in energy expenditure. Further, the FGF19 transgenic mice did not become obese or diabetic on a high fat diet. The FGF19 transgenic mice had increased brown adipose tissue mass and decreased liver expression of acetyl coenzyme A carboxylase 2, providing two mechanisms by which FGF19 may increase energy expenditure. Consistent with the reduction in expression of acetyl CoA carboxylase 2, liver triglyceride levels were reduced.

Fibroblast growth factor 19 increases metabolic rate and reverses dietary and leptin-deficient diabetes.

Hormonal control of metabolic rate can be important in regulating the imbalance between energy intake and expenditure that underlies the development of obesity. In mice fed a high-fat diet, human fibroblast growth factor 19 (FGF19) increased metabolic rate [1.53 +/- 0.06 liters O(2)/h.kg(0.75) (vehicle) vs. 1.93 +/- 0.05 liters O(2)/h.kg(0.75) (FGF19); P < 0.001] and decreased respiratory quotient [0.82 +/- 0.01 (vehicle) vs. 0.80 +/- 0.01 (FGF19); P < 0.05]. In contrast to the vehicle-treated mice that gained weight (0.14 +/- 0.05 g/mouse.d), FGF19-treated mice lost weight (-0.13 +/- 0.03 g/mouse.d; P < 0.001) without a significant change in food intake. Furthermore, in addition to a reduction in weight gain, treatment with FGF19 prevented or reversed the diabetes that develops in mice made obese by genetic ablation of brown adipose tissue or genetic absence of leptin. To explore the mechanisms underlying the FGF19-mediated increase in metabolic rate, we profiled the FGF19-induced gene expression changes in the liver and brown fat. In brown adipose tissue, chronic exposure to FGF19 led to a gene expression profile that is consistent with activation of this tissue. We also found that FGF19 acutely increased liver expression of the leptin receptor (1.8-fold; P < 0.05) and decreased the expression of acetyl coenzyme A carboxylase 2 (0.6-fold; P < 0.05). The gene expression changes were consistent with the experimentally determined increase in fat oxidation and decrease in liver triglycerides. Thus, FGF19 is able to increase metabolic rate concurrently with an increase in fatty acid oxidation.

Photosensitizers form in histidine buffer and mediate the photodegradation of a monoclonal antibody.

Fluorescent light (FL) photodegradation of a monoclonal antibody (mAb) formulated in histidine buffer is mediated by histidine-derived photosensitizers that accumulate and greatly increase with light exposure. Histidine-derived photosensitizers are the primary mediators of Trp photooxidation. FL-photodegradation requires light exposure and is pH dependent. It is significantly reduced or eliminated by buffer exchanges, by oxygen depletion, or at pH values greater than 7. Antibody-fragment MS ion counts reveal that oxidation of a single light chain Trp in CDR1 correlates with binding loss. Multiple heavy chain methionines oxidize, but poorly correlate with binding loss. Photosensitizers extracted from photo-aged histidine buffer are potent mediators of FL-photodegradation including oxidation and, to a lesser degree, fragmentation and aggregation of the mAb. These photosensitizers absorb visible light and have neutral mass of 187.1- 386.1 Da. They are also fluorescent with ex/em at 360/450 nm. When spiked into histidine or MES buffered mAb formulations they produce a concentration dependent and pronounced increase in FL-photodegradation; however, no oxidation or loss of antibody function occurs in the dark and hydrogen peroxide does not oxidize Trp. The major component is consistent with histidine oxidation to 6a-hydroxy-2-oxo-octahydro-pyrollo[2,3-d]imidazole-5-carboxylic acid. Photosensitizer levels measured in the formulation prior to light exposure, are linearly related to the FL-photodegradation observed and can predict degradation in photostability testing.

Molecular determinants of kinase pathway activation by Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand.

Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) mainly activates programmed cell death through caspases. By contrast, TNF primarily induces gene transcription through the inhibitor of kappaB kinase (IKK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. Apo2L/TRAIL also can stimulate these kinases, albeit less strongly; however, the underlying mechanisms of this stimulation and its relation to apoptosis are not well understood. Here we show that Apo2L/TRAIL activates kinase pathways by promoting the association of a secondary signaling complex, subsequent to assembly of a primary, death-inducing signaling complex (DISC). The secondary complex retained the DISC components FADD and caspase-8, but recruited several factors involved in kinase activation by TNF, namely, RIP1, TRAF2, and NEMO/IKKgamma. Secondary complex formation required Fas-associated death domain (FADD), as well as caspase-8 activity. Apo2L/TRAIL stimulation of JNK and p38 further depended on RIP1 and TRAF2, whereas IKK activation required NEMO. Apo2L/TRAIL induced secretion of interleukin-8 and monocyte chemoattractant protein-1, augmenting macrophage migration. Thus, Apo2L/TRAIL and TNF organize common molecular determinants in distinct signaling complexes to stimulate similar kinase pathways. One function of kinase stimulation by Apo2L/TRAIL may be to promote phagocytic engulfment of apoptotic cells.