Reduction of liver macrophage transduction by pseudotyping lentiviral vectors with a fusion envelope from Autographa californica GP64 and Sendai virus F2 domain

<p>Abstract</p> <p>Background</p> <p>Lentiviral vectors are well suited for gene therapy because they can mediate long-term expression in both dividing and nondividing cells. However, lentiviral vectors seem less suitable for liver gene therapy because systemically administered lentiviral vectors are preferentially sequestered by liver macrophages. This results in a reduction of available virus and might also increase the immune response to the vector and vector products.</p> <p>Reduction of macrophage sequestration is therefore essential for efficient lentiviral liver gene therapy.</p> <p>Results</p> <p>Fusions were made of <it>Autographa californica </it>GP64 and the hepatocyte specific Sendai Virus envelope proteins. Lentiviral vectors were produced with either wild type GP64, Sendai-GP64, or both wild type GP64 and Sendai-GP64 and tested <it>in vitro </it>and <it>in vivo </it>for hepatocyte and macrophage gene transfer.</p> <p>Sendai-GP64 pseudotyped vectors showed specific gene transfer to HepG2 hepatoma cells, with no detectable transduction of HeLa cervical carcinoma cells, and a decreased affinity for RAW mouse macrophages. Co-expression of wild type GP64 and Sendai-GP64 resulted in improved viral titers while retaining increased affinity for HepG2 cells.</p> <p>In vivo, the Sendai-GP64 vectors also showed decreased transduction of murine liver macrophages.</p> <p>Conclusion</p> <p>We demonstrate reduced macrophage transduction <it>in vitro </it>and <it>in vivo </it>with GP64/Sendai chimeric envelope proteins.</p

Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity

<p>Abstract</p> <p>Background</p> <p>The presence of cholesterol in the Human Immunodeficiency Virus (HIV) lipid envelop is important for viral function as cholesterol depleted viral particles show reduced infectivity. However, it is less well established whether other viral membrane lipids are also important for HIV infection.</p> <p>The ABCB4 protein is a phosphatidyl choline (PC) floppase that mediates transport of PC from the inner to the outer membrane leaflet. This property enabled us to modulate the lipid composition of HIV vectors and study the effects on membrane composition and infection efficiency.</p> <p>Results</p> <p>Virus generated in the presence of ABCB4 was enriched in PC and cholesterol but contained less sphingomyelin (SM). Viral titers were reduced 5.9 fold. These effects were not observed with an inactive ABCB4 mutant. The presence of the ABC transport inhibitor verapamil abolished the effect of ABCB4 expression on viral titers.</p> <p>The ABCB4 mediated reduction in infectivity was caused by changes in the viral particles and not by components co purified with the virus because virus made in the presence of ABCB4 did not inhibit virus made without ABCB4 in a competition assay.</p> <p>Incorporation of the envelope protein was not affected by the expression of ABCB4. The inhibitory effect of ABCB4 was independent of the viral envelope as the effect was observed with two different envelope proteins.</p> <p>Conclusion</p> <p>Our data indicate that increasing the PC content of HIV particles reduces infectivity.</p

Insulin-like growth factor binding protein 5 enhances survival of LX2 human hepatic stellate cells

ABSTRACT: BACKGROUND: Expression of insulin-like growth factor binding protein 5 (IGFBP5) is strongly induced upon activation of hepatic stellate cells and their transdifferentiation into myofibroblasts in vitro. This was confirmed in vivo in an animal model of liver fibrosis. Since IGFBP5 has been shown to promote fibrosis in other tissues, the aim of this study was to investigate its role in the progression of liver fibrosis. METHODS: The effect of IGFBP5 was studied in LX2 cells, a model for partially activated hepatic stellate cells, and in human primary liver myofibroblasts. IGFBP5 signalling was modulated by the addition of recombinant protein, by lentiviral overexpression, and by siRNA mediated silencing. Furthermore, the addition of IGF1 and silencing of the IGF1R was used to investigate the role of the IGF-axis in IGFBP5 mediated effects. RESULTS: IGFBP5 enhanced the survival of LX2 cells and myofibroblasts via a >50% suppression of apoptosis. This effect of IGFBP5 was not modulated by the addition of IGF1, nor by silencing of the IGF1R. Additionally, IGFBP5 was able to enhance the expression of established pro-fibrotic markers, such as collagen Ialpha1, TIMP1 and MMP1. CONCLUSION: IGFBP5 enhances the survival of (partially) activated hepatic stellate cells and myofibroblasts by lowering apoptosis via an IGF1-independent mechanism, and enhances the expression of profibrotic genes. Its lowered expression may, therefore, reduce the progression of liver fibrosi

Primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an immune-mediated chronic cholestatic liver disease with a slowly progressive course. Without treatment, most patients eventually develop fibrosis and cirrhosis of the liver and may need liver transplantation in the late stage of disease. PBC primarily affects women (female preponderance 9–10:1) with a prevalence of up to 1 in 1,000 women over 40 years of age. Common symptoms of the disease are fatigue and pruritus, but most patients are asymptomatic at first presentation. The diagnosis is based on sustained elevation of serum markers of cholestasis, i.e., alkaline phosphatase and gamma-glutamyl transferase, and the presence of serum antimitochondrial antibodies directed against the E2 subunit of the pyruvate dehydrogenase complex. Histologically, PBC is characterized by florid bile duct lesions with damage to biliary epithelial cells, an often dense portal inflammatory infiltrate and progressive loss of small intrahepatic bile ducts. Although the insight into pathogenetic aspects of PBC has grown enormously during the recent decade and numerous genetic, environmental, and infectious factors have been disclosed which may contribute to the development of PBC, the precise pathogenesis remains enigmatic. Ursodeoxycholic acid (UDCA) is currently the only FDA-approved medical treatment for PBC. When administered at adequate doses of 13–15 mg/kg/day, up to two out of three patients with PBC may have a normal life expectancy without additional therapeutic measures. The mode of action of UDCA is still under discussion, but stimulation of impaired hepatocellular and cholangiocellular secretion, detoxification of bile, and antiapoptotic effects may represent key mechanisms. One out of three patients does not adequately respond to UDCA therapy and may need additional medical therapy and/or liver transplantation. This review summarizes current knowledge on the clinical, diagnostic, pathogenetic, and therapeutic aspects of PBC

Targeting the ABCB4 gene to control cholesterol homeostasis

INTRODUCTION: Multidrug resistance 3 (MDR3) P-glycoprotein is a lipid floppase that is encoded by the ATP-binding cassette sub-family B member 4 (ABCB4) gene and plays a crucial role in proper bile formation by transporting phosphatidylcholine across the canalicular plasma membrane of the hepatocyte into bile. The relevance of this function is underscored by the severe pathology that develops in patients with ABCB4 deficiency. This deficiency leads to the destruction of hepatocytes and cholangiocytes by bile salts, because their cytolytic action is not reduced by formation of mixed micelles with phospholipid. AREAS COVERED: Evidence that phospholipid secretion into bile is also essential for biliary cholesterol secretion as cholesterol dissolves much better in mixed micelles of bile salts and phospholipid than in pure bile salt micelles. As a consequence, net biliary cholesterol secretion depends on the amount of phospholipid secreted and hence, the expression of ABCB4 indirectly determines biliary cholesterol output. EXPERT OPINION: It can be argued that upregulation of the ABCB4 gene expression may not only be beneficial for liver pathology in patients with partial ABCB4 deficiency, but also for the prevention of gallstone formation and optimal cholesterol disposition in a much larger populatio

Ephrin A2 receptor targeting does not increase adenoviral pancreatic cancer transduction in vivo

AIM: To generate an adenoviral vector specifically targeting the EphA2 receptor (EphA2R) highly expressed on pancreatic cancer cells in vivo

Ex-vivo evaluation of gene therapy vectors in human pancreatic (cancer) tissue slices

AIM: To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions