Efficient Adenovirus-mediated Gene Transfer Research Articles

Engineering Clustered Ligand Binding Into Nonviral Vectors: αvβ3 Targeting as an Example

The development of techniques to efficiently deliver genes using nonviral approaches can broaden the application of gene delivery in medical applications without the safety concerns associated with viral vectors. Here, we designed a clustered integrin-binding platform to enhance the efficiency and targetability of nonviral gene transfer to HeLa cells with low and high densities of alpha(v)beta(3) integrin receptors. Arg-Gly-Asp (RGD) nanoclusters were formed using gold nanoparticles functionalized with RGD peptides and used to modify the surface of DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes with attached RGD nanoclusters resulted in either 5.4- or 35-fold increase in gene transfer efficiency over unmodified polyplexes for HeLa cells with low- or high-integrin surface density, respectively. The transfection efficiency obtained with the commercially available vector jetPEI-RGD was used for comparison as a vector without clustered binding. JetPEI-RGD exhibited a 1.2-fold enhancement compared to unmodified jetPEI in cells with high densities of alpha(v)beta(3) integrin receptors. The data presented here emphasize the importance of the RGD conformational arrangement on the surface of the polyplex to achieve efficient targeting and gene transfer, and provide an approach to introduce clustering to a wide variety of nanoparticles for gene delivery.

Adenoviral-mediated gene transfer to retinal explants during development and degeneration

Naturally occurring mutations of the β subunit of the cyclic guanosine monophosphate (cGMP) phosphodiesterase (β-PDE) gene in rod photoreceptors of mice and dogs are similar to one of the inherited retinal degenerations termed retinitis pigmentosa in humans. Defects in the rod β-PDE gene leading to photoreceptor cell degeneration in retinal degenerative (rd) mice can be corrected by transfer of a wild type β-PDE gene. However, the rapid photoreceptor degeneration in this mutant makes the study of gene therapy difficult. Since the retinal degeneration is slowed in vitro, we have employed retinal explants from rd mice to study factors influencing viral transduction. Retinal explants provide a rapid, efficient method to compare the transduction efficiency of adenoviral vector-mediated reporter gene delivery at different ages in normal and rd mice. Retinal explants from postnatal day (P)2 to P28 control (C57BL/6J) and P2–P42 rd mice were exposed for 20 hr to 2·5×10 8 plaque forming units (pfu) ml −1 of adenoviral vector with a β-galactosidase (Lac Z) reporter gene (Ad-CMV-Lac Z). After incubation in vector-free media for an additional 3 days, the explants were fixed and histochemically stained for β-galactosidase to reveal Lac Z gene expression. The explants were also embedded and sectioned for light microscopic observation. Transduction efficiency was higher in rd mice than in controls on all postnatal days examined. In normal retinal explants, expression of the Lac Z gene increased from P2 to a peak around P7–P8, then decreased at subsequent ages; little transduction could be found after P17. In rd mice transduction efficiency of Ad-CMV-Lac Z increased from P2 to P7, decreased by P10 and increased again after P10. The most dramatic increase in the transduction efficiency occurred in the rd retina between P10 and P15 when Lac Z was intensely expressed throughout the retina. Microscopic examination of retinal sections revealed the types and distribution of Lac Z-positive cells responsible for the deep blue staining in the retinal whole mount. In normal and rd mice, Lac Z-positive cells were located throughout the retina. However, larger numbers of Lac Z-positive cells were present at all ages examined in retinal explants from rd mice compared to normal mice. These data indicate a difference in transduction efficiency between normal and rd mice, especially after P12, and suggest efficient adenovirus-mediated gene transfer is more attainable in developing or degenerating retina. Thus, transduction efficiency in rd mice depends on the relationship between development, maturation and the degenerative state of the photoreceptor cells.

362. Widespread and Efficient Adenovirus-Mediated Gene Transfer to the Airways by DEAE Dextran and Sodium Caprate in Adult Mice and Fetal Sheep after Minimally Invasive Tracheal Application

362. Widespread and Efficient Adenovirus-Mediated Gene Transfer to the Airways by DEAE Dextran and Sodium Caprate in Adult Mice and Fetal Sheep after Minimally Invasive Tracheal Application

Efficient adenovirus-mediated gene transfer into primary T cells and thymocytes in a new coxsackie/adenovirus receptor transgenic model

BackgroundGene transfer studies in primary T cells have suffered from the limitations of conventional viral transduction or transfection techniques. Replication-defective adenoviral vectors are an attractive alternative for gene delivery. However, naive lymphocytes are not readily susceptible to infection with adenoviruses due to insufficient expression of the coxsackie/adenovirus receptor.ResultsTo render T cells susceptible to adenoviral gene transfer, we have developed three new murine transgenic lines in which expression of the human coxsackie/adenovirus receptor (hCAR) with a truncated cytoplasmic domain (hCARΔcyt) is limited to thymocytes and lymphocytes under direction of a human CD2 mini-gene. hCARΔcyt.CD2 transgenic mice were crossed with DO11.10 T cell receptor transgenic mice (DO11.hCARΔcyt) to allow developmental studies in a defined, clonal T cell population. Expression of hCARΔcyt enabled adenoviral transduction of resting primary CD4+ T cells, differentiated effector T cells and thymocytes from DO11.hCARΔcyt with high efficiency. Expression of hCARΔcyt transgene did not perturb T cell development in these mice and adenoviral transduction of DO11.hCARΔcyt T cells did not alter their activation status, functional responses or differentiative potential. Adoptive transfer of the transduced T cells into normal recipients did not modify their physiologic localization.ConclusionThe DO11.hCARΔcyt transgenic model thus allows efficient gene transfer in primary T cell populations and will be valuable for novel studies of T cell activation and differentiation.

Highly efficient adenovirus-mediated gene transfer to cardiac myocytes after single-pass coronary delivery.

Efficient and homogeneous gene transfer to cardiac myocytes is a major target in myocardial gene therapy. The aim of this study was to determine the conditions permitting efficient, homogeneous, adenovirus-mediated gene transfer to cardiac myocytes, with a view to application during coronary artery catheterization. Gene transfer to adult rat ventricular myocytes was conducted using type 5 adenoviruses carrying the lacZ reporter gene. Adenovirus delivery via coronary arteries was performed on isolated perfused rat hearts, and gene transfer efficiency was analyzed on whole ventricles, freshly isolated myocytes, and cultured myocytes. Single-pass delivery of 1 X 10(9) PFU associated with 1 min of no-flow yielded only 1 +/- 0.5% of positive myocytes. Pretreatment by histamine perfusion (10(-5) M final concentration) increased this value to 30 +/- 9% (p < 0.001), and pretreatment by Ca2+-free buffer perfusion increased it to 67 +/- 8% (p < 0.001). Combination of the two pretreatments had no additional effect. Increasing the viral dose to 3 X 10(9) PFU increased transfection efficiency only in permeabilized vessels. The 1-min no-flow period after adenovirus delivery was crucial for efficient gene transfer: despite histamine pretreatment, only 2 +/- 1% positive myocytes were observed without flow interruption (p < 0.05 versus 1 min of no-flow). Gene transfer was shown to occur in situ during cardiac perfusion, rather than during heart digestion or myocyte isolation. This study shows that highly efficient adenovirus-mediated gene transfer to cardiac myocytes in situ can be achieved by single-pass intracoronary vector delivery, provided that vascular permeability is first increased and coronary flow is briefly interrupted.

Influence of temperature on adenovirus-mediated gene transfer.

The transfer of recombinant genes to donor organs may allow for novel therapeutic approaches to the challenges of acute and chronic rejection. Adenoviral vectors are capable of efficient gene transfer, but use of these vectors during donor organ preservation may be less efficient due to the low temperature. This study was designed to examine the effect of temperature on the efficiency of adenovirus-mediated gene transfer. Gene transfer to human endothelial cells, porcine vascular smooth muscle cells and cultured rat thoracic aortas was examined. Incubation with an adenoviral vector encoding for E. coli beta-galactosidase was performed for 1 h at three different temperatures: 4 degrees C, 10 degrees C and 37 degrees C. Transgene expression was assessed by histochemical staining for beta-galactosidase in transduced cells and by evaluation of beta-galactosidase activity in organ cultures. Both in human endothelial cells and vascular smooth muscle cells the percentage of positively staining cells following transduction at 37 degrees C was significantly greater than at 4 degrees C and at 10 degrees C (30.55 +/- 7.26% vs. 14.29 +/- 3.79% and 12.43 +/- 2.47%, respectively for endothelial cells, P < 0.01 vs. 4 degrees C and 10 degrees C; and 28.25 +/- 4.52% vs. 17.91 +/- 3.76% and 16.63 +/- 3.92%, respectively for smooth muscle cells, P < 0.05 vs. 4 degrees C, P < 0.01 vs. 10 degrees C). Beta-galactosidase activity was significantly greater in aortas transduced at 37 degrees C than in vessels transduced at 4 degrees C and 10 degrees C (289,700 +/- 113,300 vs. 149,600 +/- 54,390 and 108,800 +/- 23,140 relative chemiluminesce units/mg of total protein, respectively; P < 0.05 vs. 4 degrees C, P < 0.001 vs. 10 degrees C). The present study demonstrates that the efficiency of adenovirus-mediated gene transfer is significantly reduced at lower temperatures. The need for cold preservation of donor organs may render efficient adenovirus-mediated gene transfer more difficult in the transplantation setting.

Role of integrin expression in adenovirus-mediated gene delivery to the intestinal epithelium.

Adenoviral vectors are being developed for oral delivery of therapeutic genes to the intestine. Initial studies in the rat using mucolytics and direct application of adenovirus encoded with the interleukin-1 receptor antagonist gene to the jejunum produced limited gene expression. The goal of this study was to determine the role of integrins in adenovirus-mediated gene delivery to the intestinal epithelium. Integrins are involved in cellular differentiation and tight junction formation and mediate adenoviral internalization. Results from Caco-2 and IEC-18 cells suggest that, as enterocytes differentiate, cell-surface integrin expression decreases. Pretreatment of Caco-2 cells with RGD peptides reduced adenoviral transduction efficiency by 80% in undifferentiated cells and 20% in differentiated cells. Both differentiated and undifferentiated IEC-18 cells showed a 70% drop in transduction when pretreated with the peptide. Infection inhibition studies with monoclonal antibodies further suggest that alpha(v)beta3 and alpha6beta1 integrins play significant roles in adenoviral internalization in the intestine. Expression of integrins in cell culture models of the intestine correlated with in vivo expression in intestinal segments. These results indicate that the ileum is a prime target for efficient adenovirus-mediated gene transfer in the rat. To enhance transduction in differentiated enterocytes (probable targets for oral gene delivery), Caco-2 cells were treated with interleukin-1beta (a cytokine known to increase integrin expression) prior to administration of the virus. Transduction efficiency increased four-fold.

Isolated-organ perfusion for local gene delivery: efficient adenovirus-mediated gene transfer into the liver.

Targeted gene delivery is essential for gene therapy involving in vivo gene transfer. In the present study we analyzed the efficiency and tissue-specificity of gene transfer into the liver with recombinant adenoviruses. Adenovirus vectors carrying the E. coli lacZ gene (Ad.RSV.beta-gal) and the firefly luciferase gene (AdCMV-luc) as reporters were administered to the liver of adult Wistar rats, either via infusion into the portal vein (intraportal infusion; IPI) or via perfusion of the vascularity isolated liver (isolated liver perfusion; ILP). Ex vivo liver perfusion experiments with Ad.RSV. beta-gal were used to optimize the conditions for hepatic gene transfer. Ex vivo perfusion of rat livers with 2 x 10(9) plaque forming units (p.f.u) Ad RSV.beta-gal was sufficient to infect about 20% of the liver parenchymal cells. Perfusion with chelating agents (1 mM EGTA, or 2 mM EDTA) prior to the administration of the vector increased the efficiency to at least 40%. Similar efficiencies were obtained in experiments with liver lobes of Rhesus monkeys. In vivo administration of AdCMV-luc via ILP resulted in a significantly more efficient (P = 0.028) and also more reproducible gene transfer when compared to IPI. Although detectable in both groups, extrahepatic luciferase expression was considerably reduced in the ILP group. Our data demonstrate that IPL can be used for efficient and reproducible liver-specific gene delivery. Therefore, we think that the perfusion of vascularly isolated organs is useful as a modality for the tissue-specific administration of recombinant adenovirus vectors.

Efficient adenovirus-mediated gene transfer to basal but not columnar cells of cartilaginous airway epithelia.

Adenoviral vectors (AdV) developed for treatment of the pulmonary manifestations of cystic fibrosis (CF) can deliver, with high efficiency, transgenes to respiratory epithelial cells grown in culture. This study investigated the efficiency of AdV-mediated gene transfer to murine and human respiratory epithelium in vivo and concluded that the epithelial cells facing the lumen of the respiratory cartilaginous airways (columnar cells) are poorly transduced with AdV. Mechanical injury to the epithelium, however, leads to efficient in vivo gene transfer by exposing a susceptible epithelial subtype (basal cells). Increased gene transfer efficiency in vivo after injury is not a nonspecific response because the proliferative status of the epithelium after injury was shown not to correlate temporally to the increased transduction susceptibility of the epithelium. Although basal cells were the cell type transduced at the time of vector delivery, with time, basal cell differentiation to columnar cells occurred with maintenance of transgene expression. Collectively, these results show that murine and human cartilaginous airways are poorly transduced by AdV. To correct the cartilaginous airway CF bioelectrical defect in vivo, efforts should be directed to increase the tropism of AdV to the columnar airway epithelial cells.

Efficient adenovirus-mediated gene transfer into human cancer cell lines derived from digestive tract

The efficiency of gene transfer into human cancer cells from digestive tract was evaluated using a replication-deficient recombinant adenovirus (Ad) vector harboring a lacZ gene of E. coli as a reporter gene (AxCALacZ). Average percent X-gal staining of esophageal cancer cell lines was 46%, that of gastric cancer cell lines 82% and that of colon cancer cell lines 70% at 3 days after Ad vector infection. X-gal staining in vitro continued 2 months after infection. By the direct injection of adenovirus vector to the tumors in nude mice, a certain percentage of tumor cells was stained by the X-gal. Colon26 cell line infected with AxCALacZ was implanted in BALB/c mice immunized with AxCALacZ, and tumor growth was suppressed. We presume this was due to anti-adenoviral immunity.

Expression of alpha v beta 5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway

Recombinant adenoviruses are being evaluated for gene therapy of cystic fibrosis lung disease with the goal of reconstituting the expression of the cystic fibrosis transmembrane conductance regulator in pulmonary epithelia by direct administration of the virus into the airway. The therapeutic potential of recombinant adenoviruses is limited in part by the relative inefficiency by which gene transfer occurs. This study uses a human bronchial xenograft model to study adenovirus infection in the human airway in an attempt to define the molecular events that limit gene transfer. Our studies of the human airway confirm previous observations of cell lines that have indicated a two-step process for adenovirus entry, which begins with the binding of the virus to the cell through the fiber protein and continues with internalization via interactions among cellular integrins and an RGD motif (Arg-Gly-Asp) in the penton base. Furthermore, the level of maturity of the epithelia in xenografts has a major impact on gene transfer. Undifferentiated epithelia express high levels of alpha v beta 5 integrins and are easily infected with recombinant adenoviruses; gene transfer is completely inhibited with excess fiber and partially inhibited with RGD peptide and alpha v beta 5 integrin antibody. Pseudostratified epithelia do not express alpha v beta 5 integrin in differentiated columnar cells and are relatively resistant to adenovirus-mediated gene transfer; what little gene transfer occurs is inhibited by fiber but not by RGD peptide or alpha v beta 5 integrin antibody. These studies suggest that the expression of integrins in human airway epithelia limits the efficiency of gene transfer with recombinant adenoviruses. However, low-level gene transfer can occur in fully mature epithelia through alpha v beta 5 integrin-independent pathways.

Hepatic Function Is Preserved Following Liver-Directed, Adenovirus-Mediated Gene Transfer

Inherited and acquired disorders of the liver are attractive targets for gene therapy. Hepatic cells are susceptible targets for shuttle vectors because of a diversity of protein and viral receptors and accessibility of a selective afferent blood supply. Preservation of existing hepatic cell integrity and metabolic function is of paramount importance for successful whole animal gene therapy trials. In this report, we examine hepatic cell function and integrity following adenovirus-mediated reporter gene transfer to the liver in vivo. E1-deleted, replication-defective adenovectors encoding the LacZ gene driven by the human CMV promoter were delivered to the liver by isolated portal perfusion. The gene transfer rate, as determined by specific histochemical staining, approached 30% with recombinant protein detectable by Western blot throughout the course of study. Hepatic cell integrity as assessed by histology and hepatic enzyme profile (serum aspartate aminotransferase, γ-glutamyl transpeptidase) demonstrated normal cellular architecture and no significant difference between transfected liver and controls. Hepatic synthetic and metabolic function, as determined by albumin levels, prothrombin time, and bilirubin, were similar between the two study groups. This study demonstrates that efficient adenovirus-mediated gene transfer and expression in the rat liver do not compromise hepatic cell metabolism and integrity.

Highly efficient adenovirus-mediated gene transfer into renal cells in culture

In summary, we have shown that adenovirus vector efficiently introduced foreign gene into cultured renal cells both of mesangial and tubular origin. Genes transferred were properly expressed to produce the molecules of expected function. It was possible to introduce the gene into nearly 100% of the cells treated. Expression of the gene began as early as 12 hours after the infection, increased until 48 hours and persisted at least up to eight days. Finally, the vector was non-toxic to the cells, as judged from simple toxicity tests. Successful application of adenovirus vector enables for us to study function of pertinent molecules in suitable host cells and opens a new way for examining renal cellular physiology and pathophysiology.

Efficient Adenovirus-Mediated Gene Transfer into Human Blood Monocyte-Derived Macrophages

The efficiency of gene transfer into human blood inonocyte-derived macrophages has been evaluated using a replication-defective adenovirus vector harboring a lac Z gene of E. coli as a reporter gene. Whereas, no β-galactosidase activity was found in freshly infected purified monocytes, 40% to 80% of infected macrophages which derived from these monocytes showed a β-galactosidase activity, 2 to 4 days after infection and lasted for at least 3 weeks. Moreover, β-galactosidase activity was found in infected monocyte/macrophages 7 days after their injection into a human tumor preestablished in nude mice. These data indicate that it is possible to transfer and stably express a gene of potential therapeutic function into human monocyte-derived macrophages using an adenovirus vector.

Traditions and new trends in cell physiology

EDITORIALTraditions and new trends in cell physiologyPhilip A. KnaufPhilip A. KnaufPublished Online:01 Jul 1984https://doi.org/10.1152/ajpcell.1984.247.1.C1MoreSectionsPDF (272 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmail "Traditions and new trends in cell physiology." American Journal of Physiology-Cell Physiology, 247(1), p. C1 Back to Top Next FiguresReferencesRelatedInformationCited ByCT-Based Diagnosis of Low Vertebral Bone Mineral Density Is Associated with Hypercalciuria and Hypocitraturia on Opportunistic ImagingJournal of Endourology, Vol. 32, No. 9Increased risk of bone fracture among patients with urinary calculi: a nationwide longitudinal population-based study19 December 2014 | Osteoporosis International, Vol. 26, No. 4Acid–Base Balance and Bone Health28 November 2014Safety of carbonic anhydrase inhibitors10 March 2014 | Expert Opinion on Drug Safety, Vol. 13, No. 4Renal calculus disease4 March 2013How Permissive Should Hypercapnia Be?Local and Systemic Effects of HypercapniaThe effects of acid on boneCurrent Opinion in Nephrology and Hypertension, Vol. 9, No. 4Highly Efficient Adenovirus-Mediated Gene Transfer to Cardiac Myocytes after Single-Pass Coronary DeliveryHuman Gene Therapy, Vol. 11, No. 7Pathogenesis of Idiopathic Calcium Nephrolithiasis: Update 199729 September 2008 | Critical Reviews in Clinical Laboratory Sciences, Vol. 35, No. 2PERMEABILITY OF MYOCARDIAL CAPILLARIES TO HYDROPHILIC DRUGS: THE PARACELLULAR PATHWAYClinical and Experimental Pharmacology and Physiology, Vol. 23, No. 10-11The Pathophysiology of DiarrheaStructure and Regulation of the Amiloride-Sensitive Epithelial Sodium ChannelUrinary calcium and oxalate excretion in childrenPediatric Nephrology, Vol. 9, No. 1Famine, Fiber, Fatty Acids, and Failed Colonic Absorption: Does Fiber Fermentation Ameliorate Diarrhea?29 November 2016 | Journal of Parenteral and Enteral Nutrition, Vol. 18, No. 1Segmental differences in short-chain fatty acid transport in rabbit colon: Effect of pH and Na1 November 1993 | The Journal of Membrane Biology, Vol. 136, No. 2Structure-Function Correlations of Colon and Distal NephronThe Pathogenesis and Treatment of Kidney StonesNew England Journal of Medicine, Vol. 327, No. 16Parathyroid Function in Relation to Intestinal Function and Renal Calcium Reabsorption in Patients with Nephrolithiasis15 February 2010 | Scandinavian Journal of Urology and Nephrology, Vol. 26, No. 1The risk of nephrolithiasis with oral calciumsupplementationCalcified Tissue International, Vol. 48, No. 2Screening for HypercalciuriaPediatrics International, Vol. 32, No. 6An assessment of plasma histamine concentrations during documented endotoxic shockAgents and Actions, Vol. 31, No. 3-4Genetics of UrolithiasisEffects of Heat-Stable Escherichia coli Enterotoxin on Intestinal Alkaline Secretion and Transepithelial Potential Difference in the Rat Intestines in Vivo8 July 2009 | Scandinavian Journal of Gastroenterology, Vol. 25, No. 1Response to Exogenous 1,25(OH)2D during a Low-Calcium Diet in Normal Subjects and Idiopathic Renal-Stone FormersUrolithiasis in children: Current medical managementPediatric Nephrology, Vol. 3, No. 3Evidence for a prostaglandin-mediated bone resorptive mechanism in subjects with fasting hypercalciuriaCalcified Tissue International, Vol. 43, No. 2Methods of Studying Sympathoadrenal Activity in Man24 April 2009 | Acta Medica Scandinavica, Vol. 223, No. 6NaCl Transport in the Vertebrate IntestineIdiopathic Hypercalciuria in Children.Acta Paediatrica, Vol. 76, No. 2Peroraler Calcium-Belastungstest unter freier Diät bei idiopathischer Calcium-Nephrolithiasis — Möglichkeiten und GrenzenKlinische Wochenschrift, Vol. 64, No. 20Glucose Counterregulation, Hypoglycemia, and Intensive Insulin Therapy in Diabetes MellitusNew England Journal of Medicine, Vol. 313, No. 4Prospects for Gene Therapy for the Fibrosed Heart: Targeting Regulators of Extracellular Matrix Turnover More from this issue > Volume 247Issue 1July 1984Pages C1-C1 Copyright & PermissionsCopyright © 1984 the American Physiological Societyhttps://doi.org/10.1152/ajpcell.1984.247.1.C1History Published online 1 July 1984 Published in print 1 July 1984 PDF download Metrics Downloaded 16 times