Adeno-Associated Virus (AAV)-Mediated Gene Therapy for Disorders of Inherited and Non-Inherited Origin

Gene therapy is a novel promising approach for treating a spectrum of inherited and non-inherited disorders by delivering therapeutic genes to specific organs or tissues. Of the viral vectors that have been used to date to deliver the genes of interest, the adeno-associated viral (AAV) vector appears to be the most safe and effective vehicle and has the ability to maintain long-term gene and protein expression following a single injection of the vector. Gene therapy studies using AAV vector have shown significant progress not only in animal models but also in human gene therapy with no known pathogenicity. While success has been achieved in gene therapy using AAV vector to deliver the target genes for inherited disorders, however, clinical trials are yet to begin to see whether gene therapy has promise for treatment of non-inherited diseases. This chapter describes AAV biology, viral structure, and cell entry mechanisms, with special emphasis on AAV tissue tropism achieved by manipulating different serotypes and capsid engineering. This chapter also discusses successful application of the AAV vector for non-inherited disorders in animal models with particular reference to liver fibrosis, outlining advantages, disadvantages, and future challenges that this therapy may face

Transforming growth factor beta family expression at the bovine feto-maternal interface

<p>Abstract</p> <p>Background</p> <p>Endometrial remodelling is necessary for implantation in all mammalian species. The TGF beta super-family plays a crucial role in this event in humans and mice. However, the role of TGF beta super-family members during implantation is still unclear in ruminants. In the present study, the spacio-temporal expression of TGF beta super-family members including activin was explored in bovine trophoblasts and endometrial tissue during the peri-implantation period in order to elucidate whether it is essential for promoting cell proliferation at the implantation site.</p> <p>Methods</p> <p>Gene expression in the fetal membrane and endometrium of the gravid and non-gravid horn around Day 35 of gestation were analyzed with a custom-made oligo-microarray in cattle. The expression of activin and its related genes was also analyzed with quantitative RT-PCR. Activin-like activity in trophoblastic tissue and BT-1 cells was examined using a fibroblast cell proliferation test and Western blotting.</p> <p>Results</p> <p>The expression of various TGF beta super-family related genes including activin was detected in trophoblasts and the endometrium in cattle. The most intensive activin expression was found in the gravid horn endometrium, and rather intense expression was detected in the non-gravid trophoblastic tissue. Extracts from the fetal membrane including trophoblasts and purified activin both stimulated fibroblast proliferation effectively, and activin was immunologically detected in BT-1 cells, which have trophoblastic features.</p> <p>Conclusions</p> <p>Specific expression of the activin gene (gene name: inhibin beta A) was found in the gravid horn endometrium during peri-implantation. An activin-like molecule, which was derived from the endometrium and trophoblasts, stimulated the proliferation of fibroblast cells. These results suggested that as in other species, the activity of TGF beta super-family members including activin-like molecules plays a pivotal role in endometrial remodelling, which is an essential process in implantation and placentogenesis during the peri-implantation period in cattle.</p

Update on new aspects of the renin-angiotensin system in liver disease: clinical implications and new therapeutic options

A B S T R A C T The RAS (renin-angiotensin system) is now recognized as an important regulator of liver fibrosis and portal pressure. Liver injury stimulates the hepatic expression of components of the RAS, such as ACE (angiotensin-converting enzyme) and the AT 1 receptor [AngII (angiotensin II) type 1 receptor], which play an active role in promoting inflammation and deposition of extracellular matrix. In addition, the more recently recognized structural homologue of ACE, ACE2, is also up-regulated. ACE2 catalyses the conversion of AngII into Ang-(1-7) [angiotensin-(1-7)], and there is accumulating evidence that this &apos;alternative axis&apos; of the RAS has anti-fibrotic, vasodilatory and anti-proliferative effects, thus counterbalancing the effects of AngII in the liver. The RAS is also emerging as an important contributor to the pathophysiology of portal hypertension in cirrhosis. Although the intrahepatic circulation in cirrhosis is hypercontractile in response to AngII, resulting in increased hepatic resistance, the splanchnic vasculature is hyporesponsive, promoting the development of the hyperdynamic circulation that characterizes portal hypertension. Both liver fibrosis and portal hypertension represent important therapeutic challenges for the clinician, and there is accumulating evidence that RAS blockade may be beneficial in these circumstances. The present review outlines new aspects of the RAS and explores its role in the pathogenesis and treatment of liver fibrosis and portal hypertension

cDNA microarray analysis of bovine embryo gene expression profiles during the pre-implantation period

BACKGROUND: After fertilization, embryo development involves differentiation, as well as development of the fetal body and extra-embryonic tissues until the moment of implantation. During this period various cellular and molecular changes take place with a genetic origin, e.g. the elongation of embryonic tissues, cell-cell contact between the mother and the embryo and placentation. To identify genetic profiles and search for new candidate molecules involved during this period, embryonic gene expression was analyzed with a custom designed utero-placental complementary DNA (cDNA) microarray. METHODS: Bovine embryos on days 7, 14 and 21, extra-embryonic membranes on day 28 and fetuses on days 28 were collected to represent early embryo, elongating embryo, pre-implantation embryo, post-implantation extra-embryonic membrane and fetus, respectively. Gene expression at these different time points was analyzed using our cDNA microarray. Two clustering algorithms such as k-means and hierarchical clustering methods identified the expression patterns of differentially expressed genes across pre-implantation period. Novel candidate genes were confirmed by real-time RT-PCR. RESULTS: In total, 1,773 individual genes were analyzed by complete k-means clustering. Comparison of day 7 and day 14 revealed most genes increased during this period, and a small number of genes exhibiting altered expression decreased as gestation progressed. Clustering analysis demonstrated that trophoblast-cell-specific molecules such as placental lactogens (PLs), prolactin-related proteins (PRPs), interferon-tau, and adhesion molecules apparently all play pivotal roles in the preparation needed for implantation, since their expression was remarkably enhanced during the pre-implantation period. The hierarchical clustering analysis and RT-PCR data revealed new functional roles for certain known genes (dickkopf-1, NPM, etc) as well as novel candidate genes (AW464053, AW465434, AW462349, AW485575) related to already established trophoblast-specific genes such as PLs and PRPs. CONCLUSIONS: A large number of genes in extra-embryonic membrane increased up to implantation and these profiles provide information fundamental to an understanding of extra-embryonic membrane differentiation and development. Genes in significant expression suggest novel molecules in trophoblast differentiation

A pair of temperate sub-Neptunes transiting the star EPIC 212737443

We report the validation of a new planetary system around the K3 star EPIC 212737443 using a combination of K2 photometry, follow-up high resolution imaging and spectroscopy. The system consists of two sub-Neptune sized transiting planets with radii of 2.6R⊕, and 2.7R⊕, with orbital periods of 13.6 days and 65.5 days, equilibrium temperatures of 536 K and 316 K respectively. In the context of validated K2 systems, the outer planet has the longest precisely measured orbital period, as well as the lowest equilibrium temperature for a planet orbiting a star of spectral type earlier than M. The two planets in this system have a mutual Hill radius of ΔRH = 36, larger than most other known transiting multi-planet systems, suggesting the existence of another (possibly non-transiting) planet, or that the system is not maximally packed

Mas-related G protein-coupled receptor type D antagonism improves portal hypertension in cirrhotic rats

Splanchnic vasodilatation contributes to the development and aggravation of portal hypertension (PHT). We previously demonstrated that in cirrhosis, angiotensin‐ mediates splanchnic vasodilatation through the Mas receptor (MasR). In this study, we investigated whether the recently characterized second receptor for angiotensin‐(1–7), Mas‐related G protein‐coupled receptor type D (MrgD), contributes to splanchnic vasodilatation in cirrhotic and noncirrhotic PHT. Splanchnic vascular hemodynamic and portal pressure were determined in two rat models of cirrhotic PHT and a rat model with noncirrhotic PHT, treated with either MrgD blocker D‐Pro(7)‐Ang‐(1‐7) (D‐Pro) or MasR blocker A779. Gene and protein expression of MrgD and MasR were measured in splanchnic vessels and livers of cirrhotic and healthy rats and in patients with cirrhosis and healthy subjects. Mesenteric resistance vessels isolated from cirrhotic rats were used in myographs to study their vasodilatory properties. MrgD was up‐regulated in cirrhotic splanchnic vessels but not in the liver. In cirrhotic rats, treatment with D‐Pro but not A779 completely restored splanchnic vascular resistance to a healthy level, resulting in a 33% reduction in portal pressure. Mesenteric vessels pretreated with D‐Pro but not with A779 failed to relax in response to acetylcholine. There was no splanchnic vascular MrgD or MasR up‐regulation in noncirrhotic PHT; thus, receptor blockers had no effect on splanchnic hemodynamics. Conclusion: MrgD plays a major role in the development of cirrhotic PHT and is a promising target for the development of novel therapies to treat PHT in cirrhosis. Moreover, neither MrgD nor MasR contributes to noncirrhotic PHT

Power quality data management and reporting methodologies

Deregulation of the Australian electricity supply industry is being accompanied by state regulator requirements for explicit statements on quality of supply backed up with field survey results. The continuous monitoring and storage of every voltage waveform at a selection of key sites will add significant expenses to the associated costs in managing the electricity industry while demanding an enormous quantity of information to be processed. This thesis gives a less comprehensive approach which may be accepted as a standard and consistent method of characterising the supply using simple set of power quality indices. Also presented is a power quality data management and reporting methodology which will give useful feedback to endusers, allowing assessment of operability of equipment as well as to the regulators and utilities, for the comparison of competitive distributor performance. This is supported by a power quality surveys of selected sites within electricity utilities in Australia. The thesis gives a method of analysis that can be used to conveniently convert the collected raw data into useful knowledge, covering various types of power quality disturbances i.e. continuous or variation type and discrete or event type. Literature suggests that there has been many studies undertaken on continuous disturbance characterisation and related indices. Comprehensive standards have been developed specifying objectives to be met with standard limits for all continuous disturbance types. However, there are no generally acceptable methods for characterisation of discrete disturbances and limits are not well defined in international standards. A generalised method is proposed in this thesis to characterise discrete disturbances which is essentially based on a Disturbance Severity Indicator (DSI) proportional to the customer complaint rate. Scaled versions of the CBEMA and ITIC curves have been used to give an approximation to customer complaints. The power quality reporting methodology suggested in this thesis is a consistent approach for power quality data analysis; categorised in to Short term, Medium term and Long term reporting, giving summary indices for each individual disturbance type and a single Unified Power Quality Index (UPQI) for each site and the utility. This approach would give an assessment of power quality rapidly, by means of representative numbers without overlooking important details. These indices, which are the result of characterisation and extraction from a large volume of power quality data, are easy to assess and representative of the actual impact of the disturbances they characterise. A novel methodology is also given to define discrete disturbance limits based on statistical information collected from large scale power quality surveys performed around the world. Further, Multivariable Linear Regression (MVLR) has been used as a tool to identify hidden patterns and relationships within a large quantity of power quality data in an Australian monitoring campaign. For this, a factor analysis model has been developed using MVLR and complemented with Data Mining techniques; this model reveals the good and bad factors that influence utility power quality. Finally, the power quality data management guidelines and reporting methodologies developed have been applied to representative sites of several Australian utilities, to illustrate their ability to rank sites for power quality improvements and to rank utilities for power quality benchmarking

The role of the vagal innervation of the stomach (abomasum and pylorus) and intestine (duodenum) in insulin and oxytocin release in sheep : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand

Although mechanisms regulating nutrient partitioning and milk synthesis are not fully understood in ruminants, recent studies in lactating monogastric animals have shown that the vagus nerve modulates secretion of various hormones that are implicated in the short- and long-term control of nutrient partitioning. Therefore, the overall aim of this study was to examine the role of the vagal innervation of the GI tract on insulin and oxytocin release and milk yield in sheep. In a series of experiments described in this thesis, the effect of vagotomy was studied in ewes and wethers by comparing the responses of vagotomized animals (i.e. abomasal, pyloric, duodenal and hepatic branches sectioned) with control (sham-operated) animals. Insulin release in response to a bolus injection of glucose was studied in lactating ewes (Chapters 2 and 4) or wethers (Chapters 3 and 5). The differences in responses were not significant in the experiments described in Chapters 3 and 4. However, the release of insulin from the pancreas in response to glucose injection was significantly (P < 0.05) suppressed in the vagotomized animals used in the experiments described in Chapters 2 and 5. Moreover in the experiments with wethers (Chapter 5), insulin secretion in response to glucose bolus injection was significantly (P < 0.05) higher when administered 2 h (i.e. fed state) following feeding than 22 h (i.e. fasting state). In addition, postprandial insulin concentrations were significantly (P < 0.05) lower in the vagotomized wethers than in the sham-operated wethers, but insulin secretion in the vagotomized wethers was apparently unaffected by plane of nutrition, despite significantly (P < 0.05) higher blood glucose levels in wethers on the HP intake. The insulin concentrations were, however, higher (P < 0.05) in the control group of wethers fed on the high plane (HP) of nutrition than those fed on the low plane (LP) of nutrition (Chapter 5). Insulin was released in response to the sight and/or ingestion of food, cephalic phase insulin release (CPIR), without any significant changes in blood glucose concentrations. However, the increase in insulin concentration was significantly (P < 0.05) suppressed in both vagotomized wethers and ewes in comparison with control animals (Chapters 5 and 6). Suckling increased plasma insulin concentrations in the sham-operated ewes but not in the vagotomized ewes (Chapter 6), although the difference in the concentrations between the two groups was not statistically significant (P < 0.09). Milk and fat yields were significantly (P < 0.05) reduced for one day and two days, respectively, in the vagotomized ewes compared with those of sham-operated controls, but was restored over the next 2-3 days (Chapter 2). Milk yield was not different between the two treatment groups in the second study (Chapter 4). Suckling-associated plasma oxytocin concentrations were significantly (P < 0.01) lower in the vagotomized ewes than in the sham-operated control ewes, although the difference was not statistically significant when corrected for baseline values (Chapter 4). In the next experiment (Chapter 6), oxytocin concentrations between the two treatment groups of ewes were not significantly different. However, in this experiment, suckling caused a significant (P < 0.05) increase in oxytocin concentrations from the baseline values in the sham-operated ewes fed and suckled simultaneously but not in the vagotomized ewes fed and suckled simultaneously. Vagotomy significantly (P < 0.05) increased digestibility of dry matter and nitrogen in wethers, although food intake was not different between the two treatment groups (Chapter 3). In conclusion, the findings in wethers (Chapter 5) agree with those in lactating ewes (Chapters 2 and 4) and, indicated that the effect of vagotomy on insulin release in response to glucose injection is more apparent over a short period (i.e. 2-4 h; Chapters 2 and 5) following feeding than after a longer period (i.e. 6-22 h; Chapters 4 and 5). This suggested that the pancreatic β-cells are more sensitive soon after feeding, because the vagal inputs reaching the β-cells from the GI tract are higher due to the recent consumption of food. The finding that post-prandial insulin concentrations in the vagotomized animals of HP group were significantly reduced, despite their significantly higher blood glucose levels, provide further evidence that the vagus nerve is a major determinant for sensitizing pancreatic β-cells. Furthermore the vagal innervation of the GI tract plays a major role in CPIR, and also appears to play an important role in insulin release during suckling in sheep. The concentrations of oxytocin measured in these experiments suggest that vagotomy interferes with oxytocin secretion although differences between vagotomized and sham operated ewes were often non significant. However, the data suggest that feeding stimulates OT secretion. It is possible that the failure to achieve consistent differences in milk ejection and hence removal in these studies may have been partly masked because of the anatomical features of the mammary gland of the ewe. Finally the activity of the vagus nerve influences the digestibility of dry matter and nitrogen in sheep

Power quality survey factor analysis using multivariable linear regression (MVLR)

During the past two decades, there has been a considerable number of Power Quality (PQ) monitoring programs completed throughout the world. The information collected during these surveys can provide a detailed picture of the expected electrical environment help utilities to plan their future networks in relation to power quality performance. The mass of data gathered for a sample of sites of a large-scale power quality (PQ) survey of this nature has the potential to reveal good and bad influences on power quality if an appropriate procedure for analysis can be determined. If it is known which characteristics are more important in determining the levels of particular PQ disturbances, it could be expected that other sites with similar characteristics would also present similar PQ levels. This paper aims to assess the levels of PQ disturbances and factors that influence good or bad PQ levels in a large scale PQ survey. For this, the Australian National Power Quality Benchmark Survey data has been analysed and compared for the dominant factors in long term PQ data. In this analysis, Multivariable Linear Regression (MVLR) has been identified as a useful tool for factor analysis of complex power quality data