Impaired RNA incorporation and dimerization in live attenuated leader-variants of SIV(mac239)

BACKGROUND: The 5' untranslated region (UTR) or leader sequence of simian immunodeficiency virus (SIV(mac239)) is multifunctional and harbors the regulatory elements for viral replication, persistence, gene translation, expression, and the packaging and dimerization of viral genomic RNA (vRNA). We have constructed a series of deletions in the SIV(mac239 )leader sequence in order to determine the involvement of this region in both the packaging and dimerization of viral genomic RNA. We also assessed the impact of these deletions upon viral infectiousness, replication kinetics and gene expression in cell lines and monkey peripheral blood mononuclear cells (PBMC). RESULTS: Regions on both sides of the major splice donor (SD) were found to be necessary for the efficiency and specificity of viral genome packaging. However, stem-loop1 is critical for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the initiation site of SIV-Gag have additive effects on RNA packaging and contribute to a lesser degree to RNA dimerization. The targeted disruption of structures on both sides of the SD also severely impacts viral infectiousness, gene expression and replication in both CEMx174 cells and rhesus PBMC. CONCLUSION: In the leader region of SIV(mac239), stem-loop1 functions as the primary determinant for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the translational initiation site of SIV-Gag are classified as secondary determinants and play a role in dimerization. Collectively, these data signify a linkage between the primary encapsidation determinant of SIV(mac239 )and RNA dimerization

Restriction of HIV-1 Replication in Monocytes Is Abolished by Vpx of SIVsmmPBj

Background: Human primary monocytes are refractory to infection with the human immunodeficiency virus 1 (HIV-1) or transduction with HIV-1-derived vectors. In contrast, efficient single round transduction of monocytes is mediated by vectors derived from simian immunodeficiency virus of sooty mangabeys (SIVsmmPBj), depending on the presence of the viral accessory protein Vpx. Methods and Findings: Here we analyzed whether Vpx of SIVsmmPBj is sufficient for transduction of primary monocytes by HIV-1-derived vectors. To enable incorporation of PBj Vpx into HIV-1 vector particles, a HA-Vpr/Vpx fusion protein was generated. Supplementation of HIV-1 vector particles with this fusion protein was not sufficient to facilitate transduction of human monocytes. However, monocyte transduction with HIV-1-derived vectors was significantly enhanced after delivery of Vpx proteins by virus-like particles (VLPs) derived from SIVsmmPBj. Moreover, pre-incubation with Vpx-containing VLPs restored replication capacity of infectious HIV-1 in human monocytes. In monocytes of non-human primates, single-round transduction with HIV-1 vectors was enabled. Conclusion: Vpx enhances transduction of primary human and even non-human monocytes with HIV-1-derived vectors, only if delivered in the background of SIVsmmPBj-derived virus-like particles. Thus, for accurate Vpx function the presence of SIVsmmPBj capsid proteins might be required. Vpx is essential to overcome a block of early infection steps in primary monocytes

Stem cell therapies for cardiovascular diseases: What does the future hold?

Cardiovascular disease (CVD) is a leading cause of morbidity and mortality around the globe. In Australia, one in six people are affected by CVD and a total of 4.2 million people are suffering from heart disease, stroke or vascular disease. Coronary artery disease affects around 1.2 million Australians, many of whom develop chronic heart failure as a result of ischaemic cardiomyopathy. The management of CVD has evolved tremendously in the past three decades, but the majority of treatments are not curative. Pharmacotherapy, percutaneous coronary intervention and coronary bypass grafting are important therapeutic measures, but they are unable to repair the damaged myocytes or vascular structures. Stem-cell based therapies are designed to regenerate myocardium, and attenuate or reverse the remodelling of vascular structures; therefore they may fundamentally address the structural damage or cellular degeneration in CVD. For these reasons, there have been a significant number of preclinical and clinical studies conducted in this area in recent years.Associated Grant:This work was supported by a grant from the National Natural Science Foundation of China (No. 81270104

A paper-based scaffold for enhanced osteogenic differentiation of equine adipose-derived stem cells

© 2015, Springer Science+Business Media Dordrecht.Objectives: We investigated the applicability of single layer paper-based scaffolds for the three-dimensional (3D) growth and osteogenic differentiation of equine adipose-derived stem cells (EADSC), with comparison against conventional two-dimensional (2D) culture on polystyrene tissue culture vessels. Results: Viable culture of EADSC was achieved using paper-based scaffolds, with EADSC grown and differentiated in 3D culture retaining high cell viability (>94 %), similarly to EADSC in 2D culture. Osteogenic differentiation of EADSC was significantly enhanced in 3D culture, with Alizarin Red S staining and quantification demonstrating increased mineralisation (p < 0.0001), and an associated increase in expression of the osteogenic-specific markers alkaline phosphatase (p < 0.0001), osteopontin (p < 0.0001), and runx2 (p < 0.01). Furthermore, scanning electron microscopy revealed a spherical morphology of EADSC in 3D culture, compared to a flat morphology of EADSC in 2D culture. Conclusions: Single layer paper-based scaffolds provide an enhanced environment for the in vitro 3D growth and osteogenic differentiation of EADSC, with high cell viability, and a spherical morphology

Thermosensitive hydrogel for prolonged delivery of lentiviral vector expressing neurotrophin-3 in vitro

Strappe, P ORCiD: 0000-0003-0100-0558Background: The development of tissue engineering scaffolds for gene delivery has the potential to enhance gene transfer efficiency and safety via controlled temporal and spatial delivery. Lentiviral delivery can be carried out using the natural biopolymer thermoresponsive gel, chitosan/β-glycerol phosphate (β-GP) as a carrier. Methods: Three chitosan/β-GP scaffolds were prepared with varying concentrations of chitosan and β-GP to obtain a pH and gelation temperature suitable for in situ delivery. A lentiviral vector expressing either green fluorescent protein (Lenti GFP) or neurotrophin-3 (Lenti NT-3) was incorporated into the chitosan/β-GP scaffolds and also into collagen 0.1% w/v (control). Viral elution medium was removed at various timepoints and added to the culture medium of pre-seeded HeLa or primary dorsal root ganglia (DRG) cells, respectively. GFP gene expression was quantified using fluorescence-activated cell sorting analysis. The effect of Lenti NT-3 was analyzed by measuring DRG neurite outgrowth. Results: Collagen displayed its most significant elution of virus on day 1 and chitosan/β-GP (with a final concentration of 2.17% chitosan) on day 3. Conclusions: The system shows promise for the in situ, thermoresponsive delivery of lentiviral vectors providing long-term gene expression for therapeutic factors to treat conditions such as injury to the nervous system. © 2011 John Wiley & Sons, Ltd.Associated Grant:Health Research Board of Irelan

Mesenchymal stem cells expressing eNOS and a Cav1 mutant inhibit vascular smooth muscle cell proliferation in a rat model of pulmonary hypertension

Background: This study aimed to investigate the effect of bone marrow derived mesenchymal stem cells (rBMSCs) transduced with lentiviral vectors expressing endothelial nitric oxide synthase (eNOS) and/or a mutant caveolin-1(F92A-Cav1), on the pulmonary haemodynamics and structure in a rat model of pulmonary arterial hypertension (PAH). Methods: Pulmonary arterial hypertension was induced with monocrotaline (MCT) in 60 adult male Wistar rats prior to delivery of lentiviral vector transduced rBMSCs expressing Cav1, eNOS and/or F92A-Cav1. Changes in pulmonary haemodynamics, right ventricular hypertrophy index (RVHI), and serum nitric oxide (NO) were evaluated. Ultrastructure changes in lung tissues were observed by transmission electron microscopy. Expression of Kruppel-like factor 4 (KLF4), p53, P21, eNOS, and alpha-smooth muscle actin were evaluated by real time PCR, western blotting or immunohistochemistry. Results: Treatment of PAH rats with gene modified rBMSCs (eNOS +/- Cav1 F92A) decreased right ventricular systolic pressure and improved pulmonary haemodynamics. The protein of alpha-smooth muscle actin expression was decreased whilst KLF4, p53, P21, eNOS expression, and serum NO concentration was elevated. The survival rate of rats in the treatment groups was also improved, after 35 days of observation. Conclusion: Intravenous delivery of rBMSCs expressing eNOS/F92A-Cav1 to PAH rats inhibits pulmonary vascular smooth muscle cell proliferation, and improves pulmonary haemodynamics, vascular remodelling and short-term survival. Activation of KLF4-p53 signalling pathway may be involved in these beneficial effects

Lentiviral vector delivery of short hairpin RNA to NG2 and neurotrophin-3 promotes locomotor recovery in injured rat spinal cord

Strappe, P ORCiD: 0000-0003-0100-0558Background aims. In this study we investigated the effect of neurotrophin-3 (NT-3) and knockdown of NG2, one of the main inhibitory chondroitin sulfate proteoglycans (CSPG), in the glial scar following spinal cord injury (SCI). Methods. Short hairpin (sh) RNA were designed to target NG2 and were cloned into a lentiviral vector (LV). A LV was also constructed containing NT-3. LV expressing NT-3, shRNA to NG2 or combinations of both vectors were injected directly into contused rat spinal cords 1 week post-injury. Six weeks post-injection of LV, spinal cords were examined by histology for changes in scar size and by immunohistochemistry for changes in expression of CSPG, NT-3, astrocytes, neurons and microglia/macrophages. Motor function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale. Results. Animals that received the combination treatment of LV shNG2 and LV NT-3 showed reduced scar size. These animals also showed an increase in levels of neurons and NG2, a decrease in levels of astrocytes and a significant functional recovery as assessed using the BBB locomotor scale at 2 weeks post-treatment. Conclusions. The improvement in locomotor recovery and decrease in scar size shows the potential of this gene therapy approach as a therapeutic treatment for SCI. © 2012 Informa Healthcare