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• W1968180473 abstract "Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes with no chronic toxicity. However, a toxic acute response with potentially lethal consequences has hindered their clinical applications. Liver sinusoidal endothelial cells (LSECs) and Kupffer cells are major barriers to efficient hepatocyte transduction. Understanding the mechanisms of adenoviral vector uptake by non-parenchymal cells may allow the development of strategies aimed at overcoming these important barriers and to achieve preferential hepatocyte gene transfer with reduced toxicity. Scavenger receptors on Kupffer cells bind adenoviral particles and remove them from the circulation, thus preventing hepatocyte transduction. In the present study, we show that HDAd particles interact in vitro and in vivo with scavenger receptor-A (SR-A) and with scavenger receptor expressed on endothelial cells-I (SREC-I) and we exploited this knowledge to increase the efficiency of hepatocyte transduction by HDAd vectors in vivo through blocking of SR-A and SREC-I with specific fragments antigen-binding (Fabs). Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes with no chronic toxicity. However, a toxic acute response with potentially lethal consequences has hindered their clinical applications. Liver sinusoidal endothelial cells (LSECs) and Kupffer cells are major barriers to efficient hepatocyte transduction. Understanding the mechanisms of adenoviral vector uptake by non-parenchymal cells may allow the development of strategies aimed at overcoming these important barriers and to achieve preferential hepatocyte gene transfer with reduced toxicity. Scavenger receptors on Kupffer cells bind adenoviral particles and remove them from the circulation, thus preventing hepatocyte transduction. In the present study, we show that HDAd particles interact in vitro and in vivo with scavenger receptor-A (SR-A) and with scavenger receptor expressed on endothelial cells-I (SREC-I) and we exploited this knowledge to increase the efficiency of hepatocyte transduction by HDAd vectors in vivo through blocking of SR-A and SREC-I with specific fragments antigen-binding (Fabs). Helper-dependent adenoviral (HDAd) vectors hold tremendous potential for liver-directed gene therapy because they can mediate long-term, high-level transgene expression from transduced hepatocytes with no chronic toxicity.1Brunetti-Pierri N Ng P Helper-dependent adenoviral vectors for liver-directed gene therapy.Hum Mol Genet. 2011; 20: R7-13Crossref PubMed Scopus (63) Google Scholar Following systemic administration in mice and large animal models, Ad vector particles reach the liver through the hepatic artery; in thin-wall liver sinusoids viral particles (vp) come in contact with fenestrated liver sinusoidal endothelial cells (LSEC) and with Kupffer cells, that are liver resident macrophages protruding into the vascular space and rapidly remove blood-borne Ad particles.2Ganesan LP Mohanty S Kim J Clark KR Robinson JM Anderson CL Rapid and efficient clearance of blood-borne virus by liver sinusoidal endothelium.PLoS Pathog. 2011; 7: e1002281Crossref PubMed Scopus (98) Google Scholar,3Alemany R Suzuki K Curiel DT Blood clearance rates of adenovirus type 5 in mice.J Gen Virol. 2000; 81: 2605-2609Crossref PubMed Scopus (355) Google Scholar,4Tao N Gao GP Parr M Johnston J Baradet T Wilson JM et al.Sequestration of adenoviral vector by Kupffer cells leads to a nonlinear dose response of transduction in liver.Mol Ther. 2001; 3: 28-35Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar,5Zhang Y Chirmule N Gao GP Qian R Croyle M Joshi B et al.Acute cytokine response to systemic adenoviral vectors in mice is mediated by dendritic cells and macrophages.Mol Ther. 2001; 3: 697-707Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar This uptake of vector by the reticuloendothelial system appears to contribute to Ad-induced innate immune responses.5Zhang Y Chirmule N Gao GP Qian R Croyle M Joshi B et al.Acute cytokine response to systemic adenoviral vectors in mice is mediated by dendritic cells and macrophages.Mol Ther. 2001; 3: 697-707Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar,6Smith JS Tian J Muller J Byrnes AP Unexpected pulmonary uptake of adenovirus vectors in animals with chronic liver disease.Gene Ther. 2004; 11: 431-438Crossref PubMed Scopus (41) Google Scholar,7Lieber A He CY Meuse L Schowalter D Kirillova I Winther B et al.The role of Kupffer cell activation and viral gene expression in early liver toxicity after infusion of recombinant adenovirus vectors.J Virol. 1997; 71: 8798-8807Crossref PubMed Scopus (0) Google Scholar A thorough understanding of the molecular events and players involved in recognition and uptake of intravenously injected Ad particles by the reticuloendothelial system is an important first step for overcoming the obstacles to successful HDAd-mediated gene therapy. Such efforts are required to achieve preferential hepatocyte gene transfer and to minimize Ad-mediated acute toxic response.8Brunetti-Pierri N Palmer DJ Beaudet AL Carey KD Finegold M Ng P Acute toxicity after high-dose systemic injection of helper-dependent adenoviral vectors into nonhuman primates.Hum Gene Ther. 2004; 15: 35-46Crossref PubMed Scopus (227) Google Scholar,9Morral N O'Neal WK Rice K Leland MM Piedra PA Aguilar-Córdova E et al.Lethal toxicity, severe endothelial injury, and a threshold effect with high doses of an adenoviral vector in baboons.Hum Gene Ther. 2002; 13: 143-154Crossref PubMed Scopus (154) Google Scholar Ad vectors have shown great potential for cancer treatment and have been extensively studied in clinical trials involving over 15,000 patients.10Immonen A Vapalahti M Tyynelä K Hurskainen H Sandmair A Vanninen R et al.AdvHSV-tk gene therapy with intravenous ganciclovir improves survival in human malignant glioma: a randomised, controlled study.Mol Ther. 2004; 10: 967-972Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar,11Kirn D Clinical research results with dl1520 (Onyx-015), a replication-selective adenovirus for the treatment of cancer: what have we learned?.Gene Ther. 2001; 8: 89-98Crossref PubMed Scopus (304) Google Scholar,12Peng Z Current status of gendicine in China: recombinant human Ad-p53 agent for treatment of cancers.Hum Gene Ther. 2005; 16: 1016-1027Crossref PubMed Scopus (368) Google Scholar,13Nemunaitis J Cunningham C Buchanan A Blackburn A Edelman G Maples P et al.Intravenous infusion of a replication-selective adenovirus (ONYX-015) in cancer patients: safety, feasibility and biological activity.Gene Ther. 2001; 8: 746-759Crossref PubMed Scopus (264) Google Scholar Although some effects on tumor growth have been reported, the efficacy of intravenous administration of Ad vectors in patients with metastatic cancers has been limited, likely because of rapid vector clearance from blood by Kupffer cells.13Nemunaitis J Cunningham C Buchanan A Blackburn A Edelman G Maples P et al.Intravenous infusion of a replication-selective adenovirus (ONYX-015) in cancer patients: safety, feasibility and biological activity.Gene Ther. 2001; 8: 746-759Crossref PubMed Scopus (264) Google Scholar,14Nemunaitis J Senzer N Sarmiento S Zhang YA Arzaga R Sands B et al.A phase I trial of intravenous infusion of ONYX-015 and enbrel in solid tumor patients.Cancer Gene Ther. 2007; 14: 885-893Crossref PubMed Scopus (61) Google Scholar Therefore, understanding of the receptors involved in Ad vector uptake and strategies to avoid these receptors have potential to increase tumor transduction by Ad vectors and therefore, to improve therapeutic efficacy against cancer cells.15Koski A Rajecki M Guse K Kanerva A Ristimäki A Pesonen S et al.Systemic adenoviral gene delivery to orthotopic murine breast tumors with ablation of coagulation factors, thrombocytes and Kupffer cells.J Gene Med. 2009; 11: 966-977Crossref PubMed Scopus (29) Google Scholar Ad infection occurs through different routes, with a variety of receptors on the plasma membrane of the host cells that interact with Ad capsid proteins. Serotype 5 Ad (Ad5) binds to coxsackie and adenovirus receptor,16Shayakhmetov DM Di Paolo NC Mossman KL Recognition of virus infection and innate host responses to viral gene therapy vectors.Mol Ther. 2010; 18: 1422-1429Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar,17Di Paolo NC van Rooijen N Shayakhmetov DM Redundant and synergistic mechanisms control the sequestration of blood-born adenovirus in the liver.Mol Ther. 2009; 17: 675-684Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar integrins,16Shayakhmetov DM Di Paolo NC Mossman KL Recognition of virus infection and innate host responses to viral gene therapy vectors.Mol Ther. 2010; 18: 1422-1429Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar,17Di Paolo NC van Rooijen N Shayakhmetov DM Redundant and synergistic mechanisms control the sequestration of blood-born adenovirus in the liver.Mol Ther. 2009; 17: 675-684Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar and scavenger receptors.18Haisma HJ Kamps JA Kamps GK Plantinga JA Rots MG Bellu AR Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages.J Gen Virol. 2008; 89: 1097-1105Crossref PubMed Scopus (73) Google Scholar,19Haisma HJ Boesjes M Beerens AM van der Strate BW Curiel DT Plüddemann A et al.Scavenger receptor A: a new route for adenovirus 5.Mol Pharm. 2009; 6: 366-374Crossref PubMed Scopus (71) Google Scholar Moreover, blood-borne Ad5 are opsonized by coagulation factors, especially factor X, complement, and natural antibodies, thus affecting vector tropism.20Khare R Chen CY Weaver EA Barry MA Advances and future challenges in adenoviral vector pharmacology and targeting.Curr Gene Ther. 2011; 11: 241-258Crossref PubMed Scopus (117) Google Scholar In the liver, hepatocytes are transduced via heparan sulfate proteoglycan-mediated FX binding.17Di Paolo NC van Rooijen N Shayakhmetov DM Redundant and synergistic mechanisms control the sequestration of blood-born adenovirus in the liver.Mol Ther. 2009; 17: 675-684Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar,21Shayakhmetov DM Gaggar A Ni S Li ZY Lieber A Adenovirus binding to blood factors results in liver cell infection and hepatotoxicity.J Virol. 2005; 79: 7478-7491Crossref PubMed Scopus (355) Google Scholar,22Kalyuzhniy O Di Paolo NC Silvestry M Hofherr SE Barry MA Stewart PL et al.Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo.Proc Natl Acad Sci USA. 2008; 105: 5483-5488Crossref PubMed Scopus (284) Google Scholar In contrast, the mechanism by which Kupffer cells take up Ad particles does not require coxsackie and adenovirus receptor or integrins.23Smith JS Xu Z Tian J Stevenson SC Byrnes AP Interaction of systemically delivered adenovirus vectors with Kupffer cells in mouse liver.Hum Gene Ther. 2008; 19: 547-554Crossref PubMed Scopus (57) Google Scholar Ad uptake by LSEC is almost unknown and their role in Ad liver sequestration is controversial.2Ganesan LP Mohanty S Kim J Clark KR Robinson JM Anderson CL Rapid and efficient clearance of blood-borne virus by liver sinusoidal endothelium.PLoS Pathog. 2011; 7: e1002281Crossref PubMed Scopus (98) Google Scholar,24Merrick AF Shewring LD Sawyer GJ Gustafsson KT Fabre JW Comparison of adenovirus gene transfer to vascular endothelial cells in cell culture, organ culture, and in vivo.Transplantation. 1996; 62: 1085-1089Crossref PubMed Scopus (43) Google Scholar,25Hegenbarth S Gerolami R Protzer U Tran PL Brechot C Gerken G et al.Liver sinusoidal endothelial cells are not permissive for adenovirus type 5.Hum Gene Ther. 2000; 11: 481-486Crossref PubMed Scopus (30) Google Scholar Recently, it has been reported that administration of polyinosine (poly[I]), as well as other polyanionic ligands, into mice before Ad injection drastically reduces Ad accumulation in Kupffer cells and increases hepatocyte gene transfer.18Haisma HJ Kamps JA Kamps GK Plantinga JA Rots MG Bellu AR Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages.J Gen Virol. 2008; 89: 1097-1105Crossref PubMed Scopus (73) Google Scholar,26Xu Z Tian J Smith JS Byrnes AP Clearance of adenovirus by Kupffer cells is mediated by scavenger receptors, natural antibodies, and complement.J Virol. 2008; 82: 11705-11713Crossref PubMed Scopus (162) Google Scholar This observation has led to the hypothesis that scavenger receptor-A (SR-A), and possibly other scavenger receptors, bind intravenously injected Ad particles.19Haisma HJ Boesjes M Beerens AM van der Strate BW Curiel DT Plüddemann A et al.Scavenger receptor A: a new route for adenovirus 5.Mol Pharm. 2009; 6: 366-374Crossref PubMed Scopus (71) Google Scholar,26Xu Z Tian J Smith JS Byrnes AP Clearance of adenovirus by Kupffer cells is mediated by scavenger receptors, natural antibodies, and complement.J Virol. 2008; 82: 11705-11713Crossref PubMed Scopus (162) Google Scholar Experiments in Chinese hamster ovary cells suggested that also SREC-I bind Ad5 particles.27Khare R Reddy VS Nemerow GR Barry MA Identification of adenovirus serotype 5 hexon regions that interact with scavenger receptors.J Virol. 2012; 86: 2293-2301Crossref PubMed Scopus (66) Google Scholar Pre-incubation of Kupffer cells with knob 5 resulted in strong inhibition of Ad5 infection thus suggesting a role of the knob in virus uptake by SR-A.19Haisma HJ Boesjes M Beerens AM van der Strate BW Curiel DT Plüddemann A et al.Scavenger receptor A: a new route for adenovirus 5.Mol Pharm. 2009; 6: 366-374Crossref PubMed Scopus (71) Google Scholar However, direct evidence of in vivo interaction of the Ad vector with SR-A and SREC-I have not been provided yet. In this study, we show that HDAd particles interact in vivo with SR-A and we identified the scavenger receptor expressed on endothelial cells-I (SREC-I) as a new player involved in HDAd vector binding and uptake. In addition, we show that blocking in vivo these receptors can be exploited to increase hepatocyte transduction efficiency. Improving the therapeutic index of HDAd vector by increasing hepatocyte transduction efficiency has potential for clinical translation of liver-directed gene therapy. Towards this goal, we have investigated the effect of poly[I] at increasing hepatocyte gene transfer. With the notion that poly[I] pre-treatment improves the therapeutic index of HDAd, we have determined the minimal poly[I] dose able to increase HDAd-mediated liver expression. Previous studies have used 0.2 mg of poly[I] by intravenous injection to increase Ad-mediated liver transduction.19Haisma HJ Boesjes M Beerens AM van der Strate BW Curiel DT Plüddemann A et al.Scavenger receptor A: a new route for adenovirus 5.Mol Pharm. 2009; 6: 366-374Crossref PubMed Scopus (71) Google Scholar,26Xu Z Tian J Smith JS Byrnes AP Clearance of adenovirus by Kupffer cells is mediated by scavenger receptors, natural antibodies, and complement.J Virol. 2008; 82: 11705-11713Crossref PubMed Scopus (162) Google Scholar,28Smith JS Xu Z Byrnes AP A quantitative assay for measuring clearance of adenovirus vectors by Kupffer cells.J Virol Methods. 2008; 147: 54-60Crossref PubMed Scopus (35) Google Scholar We have tested 0.4, 0.1, 0.05, and 0.025 mg of poly[I] in comparison to 0.2 mg of poly[I] (at least n = 3 per group), injected intravenously 5 minutes before injection of 5 × 1011 viral particle (vp)/kg of an HDAd vector expressing the baboon α-fetoprotein (AFP) reporter gene under the control of a liver-specific promoter (HDAd-AFP)29Brunetti-Pierri N Ng T Iannitti DA Palmer DJ Beaudet AL Finegold MJ et al.Improved hepatic transduction, reduced systemic vector dissemination, and long-term transgene expression by delivering helper-dependent adenoviral vectors into the surgically isolated liver of nonhuman primates.Hum Gene Ther. 2006; 17: 391-404Crossref PubMed Scopus (71) Google Scholar,30Brunetti-Pierri N Stapleton GE Palmer DJ Zuo Y Mane VP Finegold MJ et al.Pseudo-hydrodynamic delivery of helper-dependent adenoviral vectors into non-human primates for liver-directed gene therapy.Mol Ther. 2007; 15: 732-740Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar in wild-type C57BL/6 mice. The serum levels of AFP measured at 7 days post-injection were similar in mice pre-dosed with 0.2 or 0.05 mg of poly[I], both being approximately fivefold higher than serum AFP measured in control mice receiving saline as pre-dosing (one-way analysis of variance (ANOVA) and post-hoc Tukey's test: P < 0.01) (Figure 1a). Therefore, we concluded that the minimal effective dose of poly[I] that increases HDAd-mediated hepatocyte transduction in mice is 0.05 mg. The dose of 0.05 mg of poly[I] is also associated with long-term transgene expression following the injection of 1 × 1011 vp/kg of HDAd-AFP (n = 3 per group; Figure 1b). Therefore, poly[I] pre-treatment does not compromise HDAd-mediated long-term transgene expression. The increased hepatocyte transduction observed as a consequence of poly[I] pre-treatment is explained by a reduction of vp uptake by Kupffer cells.18Haisma HJ Kamps JA Kamps GK Plantinga JA Rots MG Bellu AR Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages.J Gen Virol. 2008; 89: 1097-1105Crossref PubMed Scopus (73) Google Scholar To confirm this, we determined the hepatic clearance rate of vector genomes between poly[I] and saline pre-treated mice. We found that hepatic HDAd copy number in animals pre-treated with poly[I] was the same as saline pre-treated mice at 1 hour post-injection but it was approximately 2.5-fold higher (t-test: P < 0.05) at 24 and 48 hours post-injection in poly[I] pre-treated compared with saline pre-treated animals (n = 3 per group; Supplementary Figure S1). The reduced hepatic HDAd genome clearance in poly[I] pre-treated mice is consistent with increased hepatocyte transduction and reduced HDAd absorption by Kupffer cells, because while HDAd transduction does not kill hepatocytes, Kupffer cells that take up Ad are killed and quickly removed from the liver.31Manickan E Smith JS Tian J Eggerman TL Lozier JN Muller J et al.Rapid Kupffer cell death after intravenous injection of adenovirus vectors.Mol Ther. 2006; 13: 108-117Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar This finding is also consistent with immunostaining data showing less Kupffer cell uptake and killing in poly[I]-treated mice.18Haisma HJ Kamps JA Kamps GK Plantinga JA Rots MG Bellu AR Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages.J Gen Virol. 2008; 89: 1097-1105Crossref PubMed Scopus (73) Google Scholar,19Haisma HJ Boesjes M Beerens AM van der Strate BW Curiel DT Plüddemann A et al.Scavenger receptor A: a new route for adenovirus 5.Mol Pharm. 2009; 6: 366-374Crossref PubMed Scopus (71) Google Scholar Acute elevation in serum lactate dehydrogenase (LDH) is a marker of Kupffer cell killing following uptake of Ad.31Manickan E Smith JS Tian J Eggerman TL Lozier JN Muller J et al.Rapid Kupffer cell death after intravenous injection of adenovirus vectors.Mol Ther. 2006; 13: 108-117Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar Thus, we measured serum LDH shortly (30 minutes) after HDAd injection and found reduced serum LDH in poly[I] pre-treated mice compared with saline pre-treated mice, a result consistent with reduced Kupffer cell killing by Ad in poly[I] pre-treated mice (n = 3 per group; Figure 1c). Although effective at improving hepatocyte gene transfer efficiency, poly[I] lowered the threshold of HDAd-mediated acute toxicity. The increase in serum interleukin-6 was higher in animals receiving 1 × 1012 vp/kg of HDAd following pre-treatment with poly[I] compared with saline pre-treated mice (n = 5 per group; one-way ANOVA and post-hoc Tukey's test: P < 0.001) (Figure 1d). At a higher dose of 5 × 1012 vp/kg, 100% mortality occurred within the first hour post-injection for mice (n = 5) pre-treated with poly[I], while no mortality was observed in mice pre-treated with saline (n = 5). Mortality was not observed neither in mice injected with 5 × 1012 vp/kg alone nor with 0.05 mg poly[I] alone. These data are consistent with previous observations of increased mortality in mice receiving poly[I] at the higher dose of 0.2 mg in combination with an early generation Ad vector.26Xu Z Tian J Smith JS Byrnes AP Clearance of adenovirus by Kupffer cells is mediated by scavenger receptors, natural antibodies, and complement.J Virol. 2008; 82: 11705-11713Crossref PubMed Scopus (162) Google Scholar The reasons for the increased toxicity of poly[I] and HDAd are unknown. Taken together, these data indicate that poly[I] increases acute toxicity of HDAd vectors and thus, poly[I] has limited potential for clinical application aiming at improving Ad-mediated liver transduction. Nevertheless, poly[I] remains a useful tool to understand mechanisms leading to improved hepatocyte transduction and to acute toxicity. Understanding the mechanisms involved in poly[I]-mediated increase in hepatocyte transduction by Ad vectors and avoidance of Kupffer cell uptake may allow the development of strategies to achieve preferential hepatocyte gene transfer. Poly[I] is thought to block the uptake of Ad particles mediated by SR-A expressed on the Kupffer cells.19Haisma HJ Boesjes M Beerens AM van der Strate BW Curiel DT Plüddemann A et al.Scavenger receptor A: a new route for adenovirus 5.Mol Pharm. 2009; 6: 366-374Crossref PubMed Scopus (71) Google Scholar To investigate the role of SR-A in improving transduction and acute toxicity, we injected wild-type C57BL/6 mice and SR-A knockout (SR-A−/−) mice in C57BL/6 background,32de Winther MP Gijbels MJ van Dijk KW van Gorp PJ suzuki H Kodama T et al.Scavenger receptor deficiency leads to more complex atherosclerotic lesions in APOE3Leiden transgenic mice.Atherosclerosis. 1999; 144: 315-321Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar with 1 × 1012 vp/kg of HDAd-AFP (n = 3 per group). We observed a significant 2.1-fold reduction of hepatocyte transduction in SR-A−/− mice compared with wild-type mice (SR-A+/+) at 7 days post-injections (one-way ANOVA and post-hoc Tukey's test: P < 0.01; Figure 2). These results were unexpected because poly[I] is a known inhibitor of SR-A18Haisma HJ Kamps JA Kamps GK Plantinga JA Rots MG Bellu AR Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages.J Gen Virol. 2008; 89: 1097-1105Crossref PubMed Scopus (73) Google Scholar,28Smith JS Xu Z Byrnes AP A quantitative assay for measuring clearance of adenovirus vectors by Kupffer cells.J Virol Methods. 2008; 147: 54-60Crossref PubMed Scopus (35) Google Scholar and knockout of this gene was expected to mimic poly[I]-mediated inhibition of SR-A, thus resulting in increased hepatocyte transduction. We next investigated whether poly[I] would increase hepatocyte transduction in SR-A−/− mice. HDAd-mediated hepatocyte transduction in SR-A−/− mice pre-treated with 0.05 mg of poly[I] was still significantly (4.1-fold) higher than saline pre-treated SR-A−/− mice (P < 0.05; Figure 2). Based on these findings, we hypothesized that other scavenger receptor(s) besides SR-A could be inhibited by poly[I] and therefore, are involved in Ad vector uptake. This hypothesis was supported by previous data showing that Ad vector DNA content following intravenous injection of Ad is similar in the livers of wild-type and SR-A−/− mice.17Di Paolo NC van Rooijen N Shayakhmetov DM Redundant and synergistic mechanisms control the sequestration of blood-born adenovirus in the liver.Mol Ther. 2009; 17: 675-684Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar,26Xu Z Tian J Smith JS Byrnes AP Clearance of adenovirus by Kupffer cells is mediated by scavenger receptors, natural antibodies, and complement.J Virol. 2008; 82: 11705-11713Crossref PubMed Scopus (162) Google Scholar SR-A and SREC-I have been previously shown to cooperate in recognition of hepatitis C virus NS3 protein and TLR2-mediated activation of myeloid cells.33Beauvillain C Meloni F Sirard JC Blanchard S Jarry U Scotet M et al.The scavenger receptors SRA-1 and SREC-I cooperate with TLR2 in the recognition of the hepatitis C virus non-structural protein 3 by dendritic cells.J Hepatol. 2010; 52: 644-651Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar Moreover, SREC-I is expressed on both macrophages and endothelial cells34Hölzl MA Hofer J Kovarik JJ Roggenbuck D Reinhold D Goihl A et al.The zymogen granule protein 2 (GP2) binds to scavenger receptor expressed on endothelial cells I (SREC-I).Cell Immunol. 2011; 267: 88-93Crossref PubMed Scopus (36) Google Scholar,35Tamura Y Osuga J Adachi H Tozawa R Takanezawa Y Ohashi K et al.Scavenger receptor expressed by endothelial cells I (SREC-I) mediates the uptake of acetylated low density lipoproteins by macrophages stimulated with lipopolysaccharide.J Biol Chem. 2004; 279: 30938-30944Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar which are barriers to Ad-mediated hepatocyte gene transfer.1Brunetti-Pierri N Ng P Helper-dependent adenoviral vectors for liver-directed gene therapy.Hum Mol Genet. 2011; 20: R7-13Crossref PubMed Scopus (63) Google Scholar Remarkably, SREC-I was also demonstrated to increase Ad5 transduction when stably transfected in Chinese hamster ovary cells.27Khare R Reddy VS Nemerow GR Barry MA Identification of adenovirus serotype 5 hexon regions that interact with scavenger receptors.J Virol. 2012; 86: 2293-2301Crossref PubMed Scopus (66) Google Scholar Therefore, we measured the levels of expression of Srec-I and II in livers of SR-A−/− mice by real-time PCR and found that their expression was indeed increased compared with wild-type C57BL/6 mice (t-test: P < 0.01 and P < 0.05, respectively; Figure 3). As a control, SR-A levels were also measured in SR-A−/− mice and were not detectable (data not shown). Therefore, SR-A−/− mice exhibit a compensatory increase in expression of SREC-I and II. Based on these findings, we hypothesized that SREC-I and SREC-II are involved in Ad uptake in vivo and their upregulation might contribute to the reduced levels of hepatic transduction observed in SR-A−/− mice. To test the hypothesis that SRECs are involved in HDAd binding, we infected J774A.1 cells, a murine monocyte-macrophage derived cell line, with AlexaFluor-555–labeled HDAd and performed immunofluorescence staining against SR-A, SREC-I, and SREC-II followed by confocal image analysis. As shown in Figure 4a, SR-A and SREC-I were found to colocalize with fluorescent HDAd vector at the plasma membrane level in 36 and 28% of analyzed cells, respectively. No colocalization was detected with SREC-II. To rule out that vector infectivity was affected by the fluorescent labeling procedure, we measured vector genome copy number by quantitative PCR (qPCR) in J774A.1 cells infected with either AlexaFluor-555–labeled HDAd or with an unlabeled HDAd, as a control, at different multiplicity of infections (MOIs). Although the infectivity of the AlexaFluor-555–labeled HDAd was slightly reduced at higher MOIs (ranging from 27 to 35%) compared with the unlabeled HDAd (t-test: P < 0.05; Supplementary Figure S2), the reduction falls within the range of variability in infectivity among vector lots.36Suzuki M Cela R Clarke C Bertin TK Mouriño S Lee B Large-scale production of high-quality helper-dependent adenoviral vectors using adherent cells in cell factories.Hum Gene Ther. 2010; 21: 120-126Crossref PubMed Scopus (33) Google Scholar We next evaluated whether SREC-I is involved in HDAd uptake and infected J774A.1 cells with HDAd expressing LacZ (HDAd-LacZ)8Brunetti-Pierri N Palmer DJ Beaudet AL Carey KD Finegold M Ng P Acute toxicity after high-dose systemic injection of helper-dependent adenoviral vectors into nonhuman primates.Hum Gene Ther. 2004; 15: 35-46Crossref PubMed Scopus (227) Google Scholar in the presence of fragments antigen-binding (Fabs) purified from anti-SR-A antibody, anti-SREC-I antibody, and an unrelated goat IgG. As controls, J774A.1 cells were infected with either HDAd-LacZ alone or in the presence of poly[I]. We found that pre-incubation with anti-SR-A blocking Fabs resulted in a reduction of virion uptake compared with cells pre-incubated with the unrelated Fab or with phosphate-buffered saline (PBS) (one-way ANOVA and post-hoc Tukey's test: P < 0.01 versus unrelated Fab and P < 0.05 versus PBS), as shown by qPCR for HDAd vector genomes (Figure 4b). Anti-SREC-I Fab also reduced HDAd uptake, even though the results did not reach statistical significance, while the combination of anti-SR-A and anti-SREC-I Fabs showed a statistically significant inhibitory effect on HDAd uptake (P < 0.01 versus unrelated Fab and P < 0.05 versus PBS) (Figure 4b). It should be pointed out that SREC-I expression in macrophages is lower compared with SR-A, and therefore these cells are not a good model to investigate SREC-I functions.35Tamura Y Osuga J Adachi H Tozawa R Takanezawa Y Ohashi K et al.Scavenger receptor expressed by endothelial cells I (SREC-I) mediates the uptake of acetylated low density lipoproteins by macrophages stimulated with lipopolysaccharide.J Biol Chem. 2004; 279: 30938-30944Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar Pre-incubation with Fab from an unrelated goat IgG did not affect HDAd transduction (Figure 4b). C57BL/6 mice were injected intravenously with 5 × 1011 vp/kg of AlexaFluor-555–labeled HDA" @default.
• W1968180473 created "2016-06-24" @default.
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• W1968180473 date "2013-04-01" @default.
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• W1968180473 title "SR-A and SREC-I Are Kupffer and Endothelial Cell Receptors for Helper-dependent Adenoviral Vectors" @default.
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