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Accurate prediction of human clearance (CL) and volume of distribution at steady state (V

Neuroinflammation is a key contributor to the pathology of Alzheimer’s disease (AD). CD33 (Siglec-3) is a transmembrane sialic acid-binding receptor on the surface of microglial cells. CD33 is upregulated on microglial cells from post-mortem AD patient brains, and high levels of CD33 inhibit uptake and clearance of amyloid beta (Aβ) in microglial cell cultures. Furthermore, knockout of CD33 reduces amyloid plaque burden in mouse models of AD. Here, we tested whether a gene therapy strategy to reduce CD33 on microglia in AD could decrease Aβ plaque load. Intracerebroventricular injection of an adeno-associated virus (AAV) vector-based system encoding an artificial microRNA targeting CD33 (miRCD33) into APP/PS1 mice reduced CD33 mRNA and TBS-soluble Aβ40 and Aβ42 levels in brain extracts. Treatment of APP/PS1 mice with miRCD33 vector at an early age (2 months) was more effective at reducing Aβ plaque burden than intervening at later times (8 months). Furthermore, early intervention downregulated several microglial receptor transcripts (e.g. CD11c, CD47 and CD36) and pro-inflammatory activation genes (e.g. Tlr4 and Il1b). Marked reductions in the chemokine Ccl2 and the pro-inflammatory cytokine Tnfα were observed at the protein level in the brain of APP/PS1 mice treated with miRCD33 vector. Overall, our data indicate that CD33 is a viable target for AAV-based knockdown strategies to reduce AD pathology. One Sentence Summary: A gene therapy approach for Alzheimer’s disease using adeno-associated virus vector-based knockdown of CD33 reduced amyloid beta accumulation and neuroinflammation.

[4-(4-Methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-yl)pyrimidine-2-amine] (JNJ-2482272), under investigation as an anti-inflammatory agent, was orally administered to rats once daily at 60 mg/kg for 6 consecutive days. Despite high plasma exposure after single administration (C

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3–5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking. Treatment of KRASG12C-mutant cancer cells with the KRAS(G12C) inhibitor AMG 510 leads to durable response in mice, and anti-tumour activity in patients suggests that AMG 510 could be effective in patients for whom treatments are currently lacking.

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing., In-depth characterization of adeno-associated virus (AAV)-mediated CRISPR delivery is still lacking. Here, the authors show high levels of integration into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in vivo.

The article deals with the design of a method for the automatic detection of static objects in the image captured by an optical microscope. The search algorithm for static objects in the image - non-moving cilia is based on texture description methods. The texture of the image is described by statistical values, where it can be noticed that background texture, cells and cilia have different mathematical statistical parameters. Just based on the different statistical parameters of the textures, the classification for each texture parameter was done separately. As a result, the resulting classification takes into account the most predominant group to which the pixel has been assigned. The output from the algorithm is a mask, where the original image is overlayed by the obtained mask and cilia area is contoured.

Mutations in the gene encoding the peroxisomal ATP-binding cassette transporter (ABCD1) cause elevations in very long-chain fatty acids (VLCFAs) and the neurodegenerative disease adrenoleukodystrophy (ALD). In most adults, this manifests as the spinal cord axonopathy adrenomyeloneuropathy (AMN). A challenge in virus-based gene therapy in AMN is how to achieve functional gene correction to the entire spinal cord while minimizing leakage into the systemic circulation, which could contribute to toxicity. In the present study, we used an osmotic pump to deliver adeno-associated viral (AAV) vector into the lumbar cerebrospinal fluid space in mice. We report that slow intrathecal delivery of recombinant AAV serotype 9 (rAAV9) achieves efficient gene transfer across the spinal cord and dorsal root ganglia as demonstrated with two different transgenes, GFP and ABCD1. In the Abcd1(–/–) mouse, gene correction after continuous rAAV9-CBA-hABCD1 delivery led to a 20% decrease in VLCFA levels in spinal cord compared with controls. The major cell types transduced were astrocytes, vascular endothelial cells, and neurons. Importantly, rAAV9 delivered intrathecally by osmotic pump, in contrast to bolus injection, reduced systemic leakage into peripheral organs, particularly liver and heart tissue.

Time of Flight (ToF) sensors are the source of various errors, including the multi-camera interference artifact caused by the parallel scanning mode of the sensors. This paper presents the novel Importance Map Based Median filtration algorithm for interference artifacts suppression, as the potential 3D filtration method. The approach is based on the processing of multiple depth frames, using the extraction of the interference region and application of the interpolation. Considering the limitations and good functionalities of proposed algorithm, the combination with some standard methods was suggested. Performance of the algorithm was evaluated on the dataset consisting of the real-world objects with different texture and morphology against popular filtering methods based on neural networks and statistics.

The non-contact measurement by using the cameras has a high potential. The paper deals about possibilities to determine the kinematic parameters of rotating or periodically repeating actions using the stroboscopic effect. This phenomenon is recorded by a highspeed camera. Using the camera setup algorithm, the measured moving object can be optically slowed down, stopped or this object can move in the opposite direction. The whole measurement can be recorded as a snapshot. Kinematic parameters were measured on a phantom (DC motor) and the appropriate filter method was determined for results.

In our research, we use depth cameras to obtain precise 3D models of patients’ heads and faces. Visual system based on these cameras is basic element of developed screening tool for obstructive sleep apnea. Decreasing of scanning time plays key role mainly for pediatric patients and suppresses generation of motion artifacts in 3D models. Used Time of Flight sensors are relatively non-expensive solution, especially in our case (parallel usage of several cameras). To obtain accurate models, the step of camera calibration is very important. In this article, we bring detailed look on geometrical distortion (radial and tangential) of used cameras and also methods for spatial calibration of system composed from three cameras. As software tool, we used Camera Calibrator implemented in MATLAB. As result of mentioned research, we found suitable multi-camera calibration method and set of parameters related to our visual system (given topology and geometrical properties of scanning cabin). Calibration decreased average distance error (measured using Hausdorff distances) from ca. 3 mm before calibration to ca. 2 mm after calibration and made easier the registration of partial models to the resulting one. Designed visual system based on depth cameras with proper calibration and interference suppression seems to have big potential for obstructive sleep apnea screening not only in pediatrics.

To find the solutions to various technical issues in image processing we often want to be able to obtain not only color but also depth information. Information about the depth helps with a better understanding and description of the scene. As a practical matter, we aim to convert the geometrical information about the object to the digital format with the highest possible accuracy. For such a conversion there are many existing methods of creating a 3D model, e.g. photogrammetry, laser scanners. These methods provide high-quality 3D information; however, they are high-priced and often limited by the size of both scanner and object and the scanning time. As an alternative, we present an optical depth sensor that can be used excluding other emerging disadvantages with lower quality of an output model though. In the future, we consider the usage of the selected sensor for specific biomedical application: diagnostic support of obstructive sleep apnea.

Ex-vivo gene therapy using stem cells or T cells transduced by retroviral or lentiviral vectors has shown remarkable efficacy in the treatment of immunodeficiencies and cancer. However, the process is expensive, technically challenging, and not readily scalable to large patient populations, particularly in underdeveloped parts of the world. Direct in vivo gene therapy would avoid these issues, and such approaches with adeno-associated virus (AAV) vectors have been shown to be safe and efficacious in clinical trials for diseases affecting differentiated tissues such as the liver and CNS. However, the ability to transduce lymphocytes with AAV in vivo after systemic delivery has not been carefully explored. Here, we show that both standard and exosome-associated preparations of AAV8 vectors can effectively transduce a variety of immune cell populations including CD4+ T cells, CD8+ T cells, B cells, macrophages, and dendritic cells after systemic delivery in mice. We provide direct evidence of T cell transduction through the detection of AAV genomes and transgene mRNA, and show that intracellular and transmembrane proteins can be expressed. These findings establish the feasibility of AAV-mediated in vivo gene delivery to immune cells which will facilitate both basic and applied research towards the goal of direct in vivo gene immunotherapies.

Hereditary hearing loss often results from mutation of genes expressed by cochlear hair cells. Gene addition using AAV vectors has shown some efficacy in mouse models, but clinical application requires two additional advances. First, new AAV capsids must mediate efficient transgene expression in both inner and outer hair cells of the cochlea. Second, to have the best chance of clinical translation, these new vectors must also transduce hair cells in non-human primates. Here, we show that an AAV9 capsid variant, PHP.B, produces efficient transgene expression of a GFP reporter in both inner and outer hair cells of neonatal mice. We show also that AAV9-PHP.B mediates almost complete transduction of inner and outer HCs in a non-human primate. In a mouse model of Usher syndrome type 3A deafness (gene CLRN1), we use AAV9-PHP.B encoding Clrn1 to partially rescue hearing. Thus, we have identified a vector with promise for clinical treatment of hereditary hearing disorders, and we demonstrate, for the first time, viral transduction of the inner ear of a primate with an AAV vector. Keywords: inner ear, AAV, non-human primate, cochlea, hair cells, adeno-associated virus vector, hereditary deafness, gene delivery

Adeno-associated viral vectors (AAVs) have demonstrated potential in applications for neurologic disorders, and the discovery that some AAVs can cross the blood-brain barrier (BBB) after intravenous injection has further expanded these opportunities for non-invasive brain delivery. Anc80L65, a novel AAV capsid designed from in silico reconstruction of the viral evolutionary lineage, has previously demonstrated robust transduction capabilities after local delivery in various tissues such as liver, retina, or cochlea, compared with conventional AAVs. Here, we compared the transduction efficacy of Anc80L65 with conventional AAV9 in the CNS after intravenous, intracerebroventricular (i.c.v.), or intraparenchymal injections. Anc80L65 was more potent at targeting the brain and spinal cord after intravenous injection than AAV9, and mostly transduced astrocytes and a wide range of neuronal subpopulations. Although the efficacy of Anc80L65 and AAV9 is similar after direct intraparenchymal injection in the striatum, Anc80L65’s diffusion throughout the CNS was more extensive than AAV9 after i.c.v. infusion, leading to widespread EGFP expression in the cerebellum. These findings demonstrate that Anc80L65 is a highly efficient gene transfer vector for the murine CNS. Systemic injection of Anc80L65 leads to notable expression in the CNS that does not rely on a self-complementary genome. These data warrant further testing in larger animal models., Graphical Abstract

The bile salt export pump (BSEP) is expressed at the canalicular domain of hepatocytes, where it mediates the elimination of monovalent bile salts into the bile. Inhibition of BSEP is considered a susceptibility factor for drug-induced liver injury that often goes undetected during nonclinical testing. Although in vitro assays exist for screening BSEP inhibition, a reliable and specific method for confirming Bsep inhibition in vivo would be a valuable follow up to a BSEP screening strategy, helping to put a translatable context around in vitro inhibition data, incorporating processes such as metabolism, protein binding, and other exposure properties that are lacking in most in vitro BSEP models. Here, we describe studies in which methods of quantitative intravital microscopy were used to identify dose-dependent effects of two known BSEP/Bsep inhibitors, 2-[4-[4-(butylcarbamoyl)-2-[(2,4-dichlorophenyl)sulfonylamino]phenoxy]-3-methoxyphenyl]acetic acid (AMG-009) and bosentan, on hepatocellular transport of the fluorescent bile salts cholylglycyl amidofluorescein and cholyl-lysyl-fluorescein in rats. Results of these studies demonstrate that the intravital microscopy approach is capable of detecting Bsep inhibition at drug doses well below those found to increase serum bile acid levels, and also indicate that basolateral efflux transporters play a significant role in preventing cytosolic accumulation of bile acids under conditions of Bsep inhibition in rats. Studies of this kind can both improve our understanding of exposures needed to inhibit Bsep in vivo and provide unique insights into drug effects in ways that can improve our ability interpret animal studies for the prediction of human drug hepatotoxicity.

In this paper, the results of the fatigue tests of the P92 and 15CH2NMFA steels are presented and discussed. The fatigue S – N curves were obtained by means of both standard and miniature test specimens. The Small Fatigue Test (SFT) specimens were manufactured partially by machining and also by waterjet cutting. The fatigue tests were performed on Amsler 10 HFP 5100 ZWICK//Roell high-frequency pulsator at room temperature and R = 0,1 and R = -1 cycle asymmetries. The results obtained on standard and SFT specimens are compared and in case of P92 steel are also compared the two methods of SFT specimens manufacturing. In case of 15CH2NMFA steel, the results of standard and SFT specimens are in a very good agreement both within the time limit fatigue range and the fatigue limit. After taking the stress concentration of SFT specimens into consideration, the results of traditional fatigue test samples and SFT samples are similar. In case of P92 steel, the leaning branch of S-N curves is much steeper for SFT specimens and the fatigue limits lower than it could be expected after the stress concentration is taken into account. The fatigue limits for equal cycle asymmetry show the same ratio of standard to SFT fatigue limits, for R = 0,1 it is 1,31, for R = 1 it is 1,32. This very good agreement can be used for the evaluation of fatigue behaviour of P92 steel by means of SFT specimens. The comparison of the two ways of SFT samples manufacturing revealed a scatter of fatigue test results, nevertheless the fatigue limits differ by 5 MPa only, so that even the influence of the manufacturing seems to be negligible.