Adenovirus-mediated gene transfer to nonparenchymal cells in normal and injured liver

2002 ◽  
Vol 282 (3) ◽  
pp. G565-G572 ◽  
Author(s):  
Qing Yu ◽  
Loretta G. Que ◽  
Don C. Rockey
Keyword(s):  
Gene Transfer ◽  
Liver Injury ◽  
Model Systems ◽  
Transduction Efficiency ◽  
Nonparenchymal Cells ◽  
Duct Ligation ◽  
Adenoviral Transduction ◽  
Injured Liver

Adenovirus-mediated gene transfer has become an important tool with which to introduce genetic material into cells. Available data emphasize efficient adenoviral transduction of parenchymal liver cells (i.e., hepatocytes) in both in vitro and in vivo model systems, typically in normal cells. The aim of this study was to evaluate gene transfer to nonparenchymal (and parenchymal) cells of the normal and injured rat liver. Hepatocytes, stellate cells, and endothelial cells were isolated by standard methods. Liver injury was induced by bile duct ligation or carbon tetrachloride administration. Cells were transduced in vitro with an adenovirus encoding β-galactosidase (Ad.β-gal) over a range of viral titers, and transduced cells were identified by detection of X-gal. In vivo transduction efficiency was studied in normal and injured livers using cell isolation techniques. Nonparenchymal cells were transduced with greater frequency than hepatocytes at all adenoviral titers tested, both in vitro and in vivo. After liver injury, adenoviral transduction was reduced for all liver cell types compared with that for cells from normal livers (at all virus titers). Notably, transduction efficiency remained greater in nonparenchymal cells than in hepatocytes after liver injury. This work implies that, to achieve comparable gene expression in the injured liver, higher adenoviral titers may be required, an important consideration as gene therapy in disease states is considered.

scholarly journals TGR5-dependent hepatoprotection through the regulation of biliary epithelium barrier function

Gut ◽  
2019 ◽  
Vol 69 (1) ◽  
pp. 146-157 ◽  
Author(s):  
Grégory Merlen ◽  
Nicolas Kahale ◽  
Jose Ursic-Bedoya ◽  
Valeska Bidault-Jourdainne ◽  
Hayat Simerabet ◽  
...  
Keyword(s):  
Liver Injury ◽  
Barrier Function ◽  
Bile Leakage ◽  
Liver Parenchyma ◽  
Knock Out ◽  
Epithelial Barrier Function ◽  
Biliary Epithelium ◽  
Duct Ligation

ObjectiveWe explored the hypothesis that TGR5, the bile acid (BA) G-protein-coupled receptor highly expressed in biliary epithelial cells, protects the liver against BA overload through the regulation of biliary epithelium permeability.DesignExperiments were performed under basal and TGR5 agonist treatment. In vitro transepithelial electric resistance (TER) and FITC-dextran diffusion were measured in different cell lines. In vivo FITC-dextran was injected in the gallbladder (GB) lumen and traced in plasma. Tight junction proteins and TGR5-induced signalling were investigated in vitro and in vivo (wild-type [WT] and TGR5-KO livers and GB). WT and TGR5-KO mice were submitted to bile duct ligation or alpha-naphtylisothiocyanate intoxication under vehicle or TGR5 agonist treatment, and liver injury was studied.ResultsIn vitro TGR5 stimulation increased TER and reduced paracellular permeability for dextran. In vivo dextran diffusion after GB injection was increased in TGR5-knock-out (KO) as compared with WT mice and decreased on TGR5 stimulation. In TGR5-KO bile ducts and GB, junctional adhesion molecule A (JAM-A) was hypophosphorylated and selectively downregulated among TJP analysed. TGR5 stimulation induced JAM-A phosphorylation and stabilisation both in vitro and in vivo, associated with protein kinase C-ζ activation. TGR5 agonist-induced TER increase as well as JAM-A protein stabilisation was dependent on JAM-A Ser285 phosphorylation. TGR5 agonist-treated mice were protected from cholestasis-induced liver injury, and this protection was significantly impaired in JAM-A-KO mice.ConclusionThe BA receptor TGR5 regulates biliary epithelial barrier function in vitro and in vivo through an impact on JAM-A expression and phosphorylation, thereby protecting liver parenchyma against bile leakage.

scholarly journals Retroviral transduction of human progenitor cells: use of granulocyte colony-stimulating factor plus stem cell factor to mobilize progenitor cells in vivo and stimulation by Flt3/Flk-2 ligand in vitro

Blood ◽  
1996 ◽  
Vol 88 (12) ◽  
pp. 4452-4462 ◽  
Author(s):  
NJ Elwood ◽  
H Zogos ◽  
T Willson ◽  
CG Begley
Keyword(s):  
Gene Transfer ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Fold Increase ◽  
Transduction Efficiency ◽  
Granulocyte Colony Stimulating Factor ◽  
Retroviral Transduction ◽  
Stimulating Factor

The clinical application of gene transfer is hindered by the availability of the multipotential stem cells and the difficulty in obtaining efficient retroviral transduction. To assess potential means by which gene transfer into human hemopoietic stem cells might be enhanced, the retroviral transduction efficiency of human bone marrow cells (BM) or peripheral blood progenitor cells (PBPC) was compared at multiple time points after in vivo administration of granulocyte colony- stimulating factor (G-CSF). This was further compared with the transduction efficiency of cells mobilized with G-CSF plus stem cell factor (SCF) in a cohort of patients randomized to receive either one or two growth factors and with normal BM function. Using the LNL6 retrovirus, retroviral transduction efficiencies of up to 19% were observed for both PBPC and BM (n = 26 patients). There was at least a 100-fold increase in PBPC with G-CSF alone and a further 30-fold increase in the total number of progenitor cells available for retroviral transduction using the combination of SCF plus G-CSF. However, pretreatment of patients with G-CSF with or without SCF did not enhance the retroviral infectability of growth factor-mobilized progenitor cells. The effect of the growth factor, Flk-2/Flt3 ligand (FL), was also examined with respect to retroviral transduction efficiency of human progenitor cells. FL plus IL-3 in vitro increased the retroviral transduction efficiency up to eightfold compared with results observed using other combinations of cytokines tested (P < .001). These findings have clinical implications both for increasing the number of target cells for in vivo gene-marking/gene-therapy studies and improving the efficiency of gene transfer.

Ultrasound targeted gene therapy in a mouse model of prostate cancer: Evaluation of immune response.

2018 ◽  
Vol 36 (6_suppl) ◽  
pp. 120-120
Author(s):  
Flavia De Carlo ◽  
Litty Thomas ◽  
Rounak Nande ◽  
Olivia Boskovic ◽  
Gailen Marshall ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Gene Transfer ◽  
Cancer Cells ◽  
Transduction Efficiency ◽  
Acquired Immunity ◽  
Prostate Cancer Cells ◽  
Adenoviral Gene Transfer ◽  
Specific Tissue

120 Background: Gene transfer to malignant sites using human adenoviruses (hAd) has been limited because of their immunogenicity. Murine cells often lack some of the receptors needed for hAd infection; therefore, are generally non-permissive for hAd infection and replication, which limits translational studies of adenoviral gene transfer techniques. We developed a gene transfer method, which uses a combination of lipid-encapsulated perfluorocarbon microbubbles (MBs) and ultrasound (US) to shield and deliver hAds to a specific tissue bypassing the requirement of the coxsackie and adenovirus receptor (CAR). Methods: Transduction efficiency and GFP protein expression of hAd.GFP was assessed by flow cytometry and fluorescence microscopy in murine TRAMP-C2 and human DU145 prostate cancer cells. Innate and acquired immunity response was determined by ELISA and CTL assay in C57BL/6 mice bearing TRAMP-C2 syngeneic tumor grafts following injections of MBs-Ad.GFP complexes in the presence or absence of ultrasound. Results: We observed that the murine prostate cancer cells TRAMP-C2 were transduced less efficiently by hAd.GFP than the human DU145 cells. We showed in vitro that the transduction rate was increased significantly in both TRAMP-C2 and DU145 prostate cancer cells when delivering the Ad particles by a combination of MBs and US. Moreover, we observed expression of the GFP transgene in both cell lines at 48 hours and 72 hours. Lack of activation of the innate and acquired immunity was observed in vivo by quantifying IL-6 and TNF-α cytokines, and by assaying neutralizing IgG antibodies and CTLs activity, following intratumoral or intravenous injections of MBs-Ad.GFP complexes in the presence or absence of ultrasound. Conclusions: This study demonstrates the feasibility of using the TRAMP-C2 murine model of prostate adenocarcinoma to translate our ultrasound-mediated MB-Ad delivery system from the bench to the clinic. Our data provides evidence that the TRAMP-C2 prostate cancer graft model is a suitable system to study in immune competent animals the capacity of lipid-encapsulated perfluorocarbon MBs and US, to shield and deliver hAds to a site-specific tissue bypassing the requirement of specific receptors.

In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance

Blood ◽  
2006 ◽  
Vol 109 (7) ◽  
pp. 2797-2805 ◽  
Author(s):  
Brian D. Brown ◽  
Giovanni Sitia ◽  
Andrea Annoni ◽  
Ehud Hauben ◽  
Lucia Sergi Sergi ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Transgene Expression ◽  
Lentiviral Vectors ◽  
Interferon Response ◽  
Type I ◽  
Nonparenchymal Cells ◽  
Antiviral Responses ◽  
Liver Gene

AbstractLiver gene transfer is a highly sought goal for the treatment of inherited and infectious diseases. Lentiviral vectors (LVs) have many desirable properties for hepatocyte-directed gene delivery, including the ability to integrate into nondividing cells. Unfortunately, upon systemic administration, LV transduces hepatocytes relatively inefficiently compared with nonparenchymal cells, and the duration of transgene expression is often limited by immune responses. Here, we investigated the role of innate antiviral responses in these events. We show that administration of LVs to mice triggers a rapid and transient IFNαβ response. This effect was dependent on functional vector particles, and in vitro challenge of antigen-presenting cells suggested that plasmacytoid dendritic cells initiated the response. Remarkably, when LVs were administered to animals that lack the capacity to respond to IFNαβ, there was a dramatic increase in hepatocyte transduction, and stable transgene expression was achieved. These findings indicate that, even in the setting of acute delivery of replication-defective vectors, IFNs effectively interfere with transduction in a cell-type–specific manner. Moreover, because disabling a single component of the innate/immune network was sufficient to establish persistent xenoantigen expression, our results raise the hope that the immunologic barriers to gene therapy are less insurmountable than expected.

scholarly journals Effect of Host Modification and Age on Airway Epithelial Gene Transfer Mediated by a Murine Leukemia Virus-Derived Vector

1998 ◽  
Vol 72 (11) ◽  
pp. 8861-8872 ◽  
Author(s):  
Larry G. Johnson ◽  
Jennifer P. Mewshaw ◽  
Hong Ni ◽  
Theodore Friedmann ◽  
Richard C. Boucher ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Brdu Incorporation ◽  
Transduction Efficiency ◽  
Airway Epithelial ◽  
Murine Leukemia ◽  
Airway Cells

ABSTRACT To study retroviral gene transfer to airway epithelia, we used a transient transfection technique to generate high titers (∼109 infectious units/ml after concentration) of murine leukemia virus (MuLV)-derived vectors pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G). Transformed (CFT1) and primary airway epithelial cells were efficiently transduced by a VSV-G-pseudotyped lacZ vector (HIT-LZ) in vitro. CFT1 cells and primary cystic fibrosis (CF) airway cell monolayers infected with a vector (HIT-LCFSN) containing human CF transmembrane conductance regulator (CFTR) in the absence of selection expressed CFTR, as assessed by Western blot analysis, and exhibited functional correction of CFTR-mediated Cl− secretion. In vitro studies of persistence suggested that pseudotransduction was not a significant problem with our vector preparations. In a sulfur dioxide (SO2) inhalational injury model, bromodeoxyuridine (BrdU) incorporation rates were measured and found to exceed 50% in SO2-injured murine tracheal epithelium. HIT-LZ vector (multiplicity of infection of ∼10) instilled into the SO2-injured tracheas of anesthetized mice transduced 6.1% ± 1.3% of superficial airway cells in tracheas of weanling mice (3 to 4 weeks old; n = 10), compared to 1.4 ± 0.9% in mice 5 weeks of age (n = 4) and 0.2% in mice older than 6 weeks (n = 15). No evidence for gene transfer following delivery of HIT-LZ to tracheas of either weanling or older mice not injured with SO2 was detected. Because only a small fraction of BrdU-labeled airway cells were transduced, we examined the stability of the vector. No significant loss of vector infectivity over intervals (2 h) paralleling those of in vivo protocols was detected in in vitro assays using CFT1 cells. In summary, high-titer vectors permitted complementation of defective CFTR-mediated Cl− transport in CF airway cells in vitro without selection and demonstrated that the age of the animal appeared to be a major factor affecting in vivo retroviral transduction efficiency.

Benchmark Concentrations for Untargeted Metabolomics vs. Transcriptomics for Liver Injury Compounds in in Vitro Liver Models

2021 ◽  
Author(s):  
David M Crizer ◽  
Sreenivasa C Ramaiahgari ◽  
Stephen S Ferguson ◽  
Julie R Rice ◽  
Paul E Dunlap ◽  
...  
Keyword(s):  
Liver Injury ◽  
Human Liver ◽  
Culture Media ◽  
Model Systems ◽  
Resolving Power ◽  
Untargeted Metabolomics ◽  
Metabolomics Data ◽  
Biological Responses

Abstract Interpretation of untargeted metabolomics data from both in vivo and physiologically-relevant in vitro model systems continues to be a significant challenge for toxicology research. Potency-based modeling of toxicological responses has served as a pillar of interpretive context and translation of testing data. In this study, we leverage the resolving power of concentration-response modeling through benchmark concentration (BMC) analysis to interpret untargeted metabolomics data from differentiated cultures of HepaRG cells exposed to a panel of reference compounds and integrate data in a potency-aligned framework with matched transcriptomic data. For this work, we characterized biological responses to classical human liver injury compounds and comparator compounds, known to not cause liver injury in humans, at 10 exposure concentrations in spent culture media by untargeted liquid chromatography-mass spectrometry (LCMS) analysis. The analyte features observed (with limited metabolites identitfied) were analyzed using BMC modeling to derive compound-induced points of departure. The results revealed liver injury compounds produced concentration-related increases in metabolomic response compared to those rarely associated with liver injury (i.e., sucrose, potassium chloride (KCl)). Moreover, the distributions of altered metabolomic features were largely comparable with those observed using high throughput transcriptomics, which were further extended to investigate the potential for in vitro observed biological responses to be observed in humans with exposures at therapeutic doses. These results demonstrate the utility of BMC modeling of untargeted metabolomics data as a sensitive and quantitative indicator of human liver injury potential.

scholarly journals Canine Adenovirus Vectors for Lung-Directed Gene Transfer: Efficacy, Immune Response, and Duration of Transgene Expression Using Helper-Dependent Vectors

2006 ◽  
Vol 80 (3) ◽  
pp. 1487-1496 ◽  
Author(s):  
Anne Keriel ◽  
Céline René ◽  
Chad Galer ◽  
Joseph Zabner ◽  
Eric J. Kremer
Keyword(s):  
Gene Transfer ◽  
Respiratory Tract ◽  
Neutralizing Antibodies ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Adenovirus Type ◽  
Transduction Efficiency

ABSTRACT A major hurdle to the successful clinical use of some viral vectors relates to the innate, adaptive, and memory immune responses that limit the efficiency and duration of transgene expression. Some of these drawbacks may be circumvented by using vectors derived from nonhuman viruses such as canine adenovirus type 2 (CAV-2). Here, we evaluated the potential of CAV-2 vectors for gene transfer to the respiratory tract. We found that CAV-2 transduction was efficient in vivo in the mouse respiratory tract, and ex vivo in well-differentiated human pulmonary epithelia. Notably, the in vivo and ex vivo efficiency was poorly inhibited by sera from mice immunized with a human adenovirus type 5 (HAd5, a ubiquitous human pathogen) vector or by human sera containing HAd5 neutralizing antibodies. Following intranasal instillation in mice, CAV-2 vectors also led to a lower level of inflammatory cytokine secretion and cellular infiltration compared to HAd5 vectors. Moreover, CAV-2 transduction efficiency was increased in vitro in human pulmonary cells and in vivo in the mouse respiratory tract by FK228, a histone deacetylase inhibitor. Finally, by using a helper-dependent CAV-2 vector, we increased the in vivo duration of transgene expression to at least 3 months in immunocompetent mice without immunosuppression. Our data suggest that CAV-2 vectors may be efficient and safe tools for long-term clinical gene transfer to the respiratory tract.

scholarly journals Retroviral transduction and expression of the human alkyltransferase cDNA provides nitrosourea resistance to hematopoietic cells

Blood ◽  
1995 ◽  
Vol 85 (11) ◽  
pp. 3342-3351 ◽  
Author(s):  
JA Allay ◽  
LL Dumenco ◽  
ON Koc ◽  
L Liu ◽  
SL Gerson
Keyword(s):  
Gene Transfer ◽  
High Efficiency ◽  
Hematopoietic Cells ◽  
K562 Cells ◽  
Fold Increase ◽  
Transduction Efficiency ◽  
Retroviral Transduction ◽  
Alkyl Moiety

Myelosuppression is the dose-limiting toxicity for nitrosourea chemotherapy. This toxicity predominantly involves modification of the O6 position of guanine with an alkyl moiety. The enzyme responsible for repair of O6-alkylguanine adducts, O6-alkylguanine-DNA alkyltransferase (alkyltransferase), is expressed at low levels in bone marrow (BM) cells. High alkyltransferase expression prevents the cytotoxicity and carcinogenicity of nitrosoureas in several transgenic and in vitro gene transfer models. We used gene transfer using a novel myeloproliferative sarcoma virus (MPSV) based retrovirus (vM5MGMT) to express the human alkyltransferase cDNA (MGMT) in human and murine hematopoietic cells. Transduced K562 cells had very high levels of alkyltransferase expression and significantly increased resistance to 1,3-bis (2-chloroethyl) nitrosourea (BCNU) as compared with untransduced K562 cells. Primary murine BM progenitors showed a high transduction efficiency with vM5MGMT and have increased BCNU resistance in vitro. After BM transplantation with vM5MGMT-transduced BM cells and BCNU treatment of these mice, BM, spleen and thymus had a 10- to 40-fold increase in alkyltransferase expression that persisted for at least 23 weeks posttransplantation. Progenitor cells procured from mice expressing high levels of alkyltransferase also had increased resistance to BCNU. Thus, an MPSV-based retroviral vector transduces mouse and human hematopoietic cells at high efficiency and results in high levels of gene expression both in vitro and in vivo. Overexpression of the alkyltransferase protein may protect hematopoietic progenitors from nitrosourea-induced myelosuppression.

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