Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps

Recombinant adeno-associated virus (rAAV) vectors are increasingly being used for clinical gene transfer and have shown great potential for the treatment of several monogenic disorders. However, contaminant DNA from producer plasmids can be packaged into rAAV alongside the intended expression cassette-containing vector genome. The consequences of this are unknown. Our analysis of rAAV preps revealed abundant contaminant sequences upstream of the AAV replication (Rep) protein driving promoter, P5, on the Rep-Cap producer plasmid. Characterization of P5-associated contaminants after infection showed transfer, persistence, and transcriptional activity in AAV-transduced murine hepatocytes, in addition to in vitro evidence suggestive of integration. These contaminants can also be efficiently translated and immunogenic, revealing previously unrecognized side effects of rAAV-mediated gene transfer. P5-associated contaminant packaging and activity were independent of an inverted terminal repeat (ITR)-flanked vector genome. To prevent incorporation of these potentially harmful sequences, we constructed a modified P5-promoter (P5-HS), inserting a DNA spacer between an Rep binding site and an Rep nicking site in P5. This prevented upstream DNA contamination regardless of transgene or AAV serotype, while maintaining vector yield. Thus, we have constructed an rAAV production plasmid that improves vector purity and can be implemented across clinical rAAV applications. These findings represent new vector safety and production considerations for rAAV gene therapy

Separation, characterisation and biological evaluation of the individual isomers of the rat selective toxicant norbormide – isolated using a chemical derivatization strategy

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB, 1), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but relatively harmless to other rodents/mammals. However, as an acute vasoactive, NRB has a rapid onset of action, often leading to sub-lethal uptake/bait shyness. Recently, it was brought to our attention that baits containing two independently sourced batches of NRB (which differed noticeably in their stereochemical composition) displayed markedly different palatability/efficacy profiles in rats. Accordingly, with a view to independently evaluating the individual isomers of NRB in rats by means of a palatability and efficacy bait trial, this research describes the isolation of the individual isomers of endo-NRB (Y, V, W and U) from the parent mixture, by means of a chemical derivatization strategy

Strategies for Inhibiting Advanced Glycation Endproduct (Age) Induced Vascular Calcification in a Smooth Muscle Cell Culture Model

Vascular calcification is implicated in a range of cardiovascular disease mechanisms, leading to an associated increase in morbidity and mortality. One such trigger are advanced glycation endproducts (AGEs), the tissue accumulation of which increases with age and is more prevalent in diabetic subjects due to oxidative stress and poor glycaemic control. The aim of this study was to investigate the osteogenic potential of AGEs and elucidate mechanisms of inhibiting these processes in a smooth muscle cell (SMC) culture model. Osteogenic differentiation of SMCs was induced using β-glycerophosphate (β-GP), carboxymethyllysine (CML), carboxyethyllysine (CEL) methylglyoxal (MGO) and glycated low density lipoprotein (gly-LDL). The cells were subsequently treated with aminoguanidine (AG), an inhibitor of AGE formation, and novel glycomimetic compounds in order to determine their anti-calcification potential in vitro using qPCR, ELISA, Alkaline phosphatase (ALP) activity and Alizarin red staining. Gly-LDL (10 µg/ml) and CML (2.5nM) increased the level of calcification observed compared to the β-GP (5 mM) positive control after 21 days (p < 0.05), with gly-LDL induced calcification apparent after 14 days. Both AG (250 µM) and the novel glycomimetic compounds reduced the level of mineralisation observed at 21 days compared with osteogenic treatments (p < 0.05). CEL (2.5 nM) and MGO (0.1 mM) both induced calcification, however mineralization was not as extensive as with β-GP. When compared to the structure of CML, the side-chain of CEL contains an extra methyl group, suggesting this group impacts RAGE receptor binding. It was also shown that β-GP combined with increased glucose concentration induced more extensive calcification unlike low glucose levels and β-GP alone. ALP activity, when stimulated with β-GP, CML and gly-LDL was greater at day 4 than at day 7, with AG reducing ALP activity measurements at day 4. Gly-LDL increases gene expression of OCN at day 4 compared with β-GP and CML, however this was reduced at day 7, corresponding with an increased expression of OPN and OPG. NOTCH-3 gene expression was also reduced at day 7. Gene expression of OPN, OPG and NOTCH-3 were reduced at both day 4 and day 7 compared with osteogenic treatments (β-GP, CML and gly-LDL). In summary, we conclude that gly-LDL and CML are potent inducers of calcification compared with β-GP, and that their osteogenic potential can be modulated by both AG and novel glycomimetic compounds

Molecular signature for receptor engagement in the metabolic peptide hormone amylin

The pancreatic peptide hormone, amylin, plays a critical role in the control of appetite, and synergizes with other key metabolic hormones such as glucagon-like peptide 1 (GLP-1). There is opportunity to develop potent and long-acting analogs of amylin or hybrids between these and GLP-1 mimetics for treating obesity. To achieve this, interrogation of how the 37 amino acid amylin peptide engages with its complex receptor system is required. We synthesized an extensive library of peptides to profile the human amylin sequence, determining the role of its disulfide loop, amidated C-terminus and receptor “capture” and “activation” regions in receptor signaling. We profiled four signaling pathways with different ligands at multiple receptor subtypes, in addition to exploring selectivity determinants between related receptors. Distinct roles for peptide sub-regions in receptor binding and activation were identified, resulting in peptides with greater activity than the native sequence. Enhanced peptide activity was preserved in the brainstem, the major biological target for amylin. Interpretation of our data using full-length active receptor models supported by molecular dynamics, metadynamics and supervised molecular dynamics simulations guided the synthesis of a potent dual agonist of GLP-1 and amylin receptors. The data offer new insights into the function of peptide amidation, how allostery drives peptide-receptor interactions, and provide a valuable resource for the development of novel amylin agonists for treating diabetes and obesity

Colocalization of connexin 36 and corticotropin-releasing hormone in the mouse brain

<p>Abstract</p> <p>Background</p> <p>Gap junction proteins, connexins, are expressed in most endocrine and exocrine glands in the body and are at least in some glands crucial for the hormonal secretion. To what extent connexins are expressed in neurons releasing hormones or neuropeptides from or within the central nervous system is, however, unknown. Previous studies provide indirect evidence for gap junction coupling between subsets of neuropeptide-containing neurons in the paraventricular nucleus (PVN) of the hypothalamus. Here we employ double labeling and retrograde tracing methods to investigate to what extent neuroendocrine and neuropeptide-containing neurons of the hypothalamus and brainstem express the neuronal gap junction protein connexin 36.</p> <p>Results</p> <p>Western blot analysis showed that connexin 36 is expressed in the PVN. In bacterial artificial chromosome transgenic mice, which specifically express the reporter gene Enhanced Green Fluorescent Protein (EGFP) under the control of the connexin 36 gene promoter, EGFP expression was detected in magnocellular (neuroendocrine) and in parvocellular neurons of the PVN. Although no EGFP/connexin36 expression was seen in neurons containing oxytocin or vasopressin, EGFP/connexin36 was found in subsets of PVN neurons containing corticotropin-releasing hormone (CRH), and in somatostatin neurons located along the third ventricle. Moreover, CRH neurons in brainstem areas, including the lateral parabrachial nucleus, also expressed EGFP/connexin 36.</p> <p>Conclusion</p> <p>Our data indicate that connexin 36 is expressed in subsets of neuroendocrine and CRH neurons in specific nuclei of the hypothalamus and brainstem.</p

Neuropeptidomics of the Supraoptic Rat Nucleus

The mammalian supraoptic nucleus (SON) is a neuroendocrine center in the brain regulating a variety of physiological functions. Within the SON, peptidergic magnocellular neurons that project to the neurohypophysis (posterior pituitary) are involved in controlling osmotic balance, lactation, and parturition, partly through secretion of signaling peptides such as oxytocin and vasopressin into the blood. An improved understanding of SON activity and function requires identification and characteriza-tion of the peptides used by the SON. Here, small-volume sample preparation approaches are optimized for neuropeptidomic studies of isolated SON samples ranging from entire nuclei down to single magnocellular neurons. Unlike most previous mammalian peptidome studies, tissues are not im-mediately heated or microwaved. SON samples are obtained from ex vivo brain slice preparations via tissue punch and the samples processed through sequential steps of peptide extraction. Analyses of the samples via liquid chromatography mass spectrometry and tandem mass spectrometry result in the identification of 85 peptides, including 20 unique peptides from known prohormones. As the sample size is further reduced, the depth of peptide coverage decreases; however, even from individually isolated magnocellular neuroendocrine cells, vasopressin and several other peptides are detected

Cardiac regeneration: different cells same goal

Cardiovascular diseases are the leading cause of mortality, morbidity, hospitalization and impaired quality of life. In most, if not all, pathologic cardiac ischemia ensues triggering a succession of events leading to massive death of cardiomyocytes, fibroblast and extracellular matrix accumulation, cardiomyocyte hypertrophy which culminates in heart failure and eventually death. Though current pharmacological treatment is able to delay the succession of events and as a consequence the development of heart failure, the only currently available and effective treatment of end-stage heart failure is heart transplantation. However, donor heart availability and immunorejection upon transplantation seriously limit the applicability. Cardiac regeneration could provide a solution, making real a dream of both scientist and clinician in the previous century and ending an ongoing challenge for this century. In this review, we present a basic overview of the various cell types that have been used in both the clinical and research setting with respect to myocardial differentiation

Long-term outcome of chronic dialysis in children

As the prevalence of children on renal replacement therapy (RRT) increases world wide and such therapy comprises at least 2% of any national dialysis or transplant programme, it is essential that paediatric nephrologists are able to advise families on the possible outcome for their child on dialysis. Most children start dialysis with the expectation that successful renal transplantation is an achievable goal and will provide the best survival and quality of life. However, some will require long-term dialysis or may return intermittently to dialysis during the course of their chronic kidney disease (CKD). This article reviews the available outcome data for children on chronic dialysis as well as extrapolating data from the larger adult dialysis experience to inform our paediatric practice. The multiple factors that may influence outcome, and, particularly, those that can potentially be modified, are discussed