Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins (original) (raw)
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Adenovirus Serotype 5 Fiber Shaft Influences In Vivo Gene Transfer in Mice
Human Gene Therapy, 2003
To assess these receptors in vivo, we mutated amino acid residues in fiber and penton that are involved in receptor interaction and showed that CAR and integrins play a minor role in hepatic transduction but that integrins can influence gene delivery to other tissues. These data suggest that an alternative entry pathway exists for hepatocyte transduction in vivo that is more important than CAR or integrins. In vitro data suggest a role for heparan sulfate glycosaminoglycans (HSG) in adenovirus transduction. The role of the fiber shaft in liver uptake was examined by introducing specific amino acid changes into a putative HSG-binding motif contained within the shaft or by preparing fiber shaft chimeras between Ad5 and Ad35 fibers. Results were obtained that demonstrate that the Ad5 fiber shaft can influence gene transfer both in vitro and to the liver in vivo. These observations indicate that the currently accepted two-step entry pathway, which involves CAR and integrins, described for adenoviral infection in vitro, is not used for hepatic gene transfer in vivo. In contrast, a v integrins influence gene delivery to the lung, spleen, heart, and kidney. The detargeted vector constructs described here may provide a foundation for the development of targeted adenoviral vectors.
Journal of Virology, 2001
The utility of adenovirus (Ad) vectors for gene therapy is restricted by their inability to selectively transduce disease-affected tissues. This limitation may be overcome by the derivation of vectors capable of interacting with receptors specifically expressed in the target tissue. Previous attempts to alter Ad tropism by genetic modification of the Ad fiber have had limited success due to structural conflicts between the fiber and the targeting ligand. Here we present a strategy to derive an Ad vector with enhanced targeting potential by a radical replacement of the fiber protein in the Ad capsid with a chimeric molecule containing a heterologous trimerization motif and a receptor-binding ligand. Our approach, which capitalized upon the overall structural similarity between the human Ad type 5 (Ad5) fiber and bacteriophage T4 fibritin proteins, has resulted in the generation of a genetically modified Ad5 incorporating chimeric fiber-fibritin proteins targeted to artificial receptor molecules. Gene transfer studies employing this novel viral vector have demonstrated its capacity to efficiently deliver a transgene payload to the target cells in a receptor-specific manner.
Gene Therapy, 2004
Adenovirus (Ad)-mediated gene delivery is a promising approach for genetic manipulation of the vasculature and is being used in both preclinical models and clinical trials. However, safety concerns relating to infection of nontarget tissue and the poor infectivity of vascular cells compared to other cell types necessitates Ad vector refinement. Here, we combine a transductional targeting approach to improve vascular cell infectivity through RGD peptide insertion into adenovirus fibers, combined with transcriptional targeting to endothelial cells using a E1 kb fragment of the fms-like tyrosine kinase receptor-1 (FLT-1) promoter. Single-and double-modified vectors were characterized in human cell lines that either support or have silenced FLT-1 expression.
Human Gene Therapy, 2004
To assess the possibilities of retargeting adenovirus to activated endothelial cells, we conjugated bifunctional polyethylene glycol (PEG) onto the adenoviral capsid to inhibit the interaction between viral knob and coxsackie-adenovirus receptor (CAR). Subsequently, we introduced an ␣v integrin-specific RGD peptide or E-selectin-specific antibody to the other functional group of the PEG molecule for the retargeting of the adenovirus to activated endothelial cells. In vitro studies showed that this approach resulted in the elimination of transgene transfer into CAR-positive cells, while at the same time specific transgene transfer to activated endothelial cells was achieved. PEGylated, retargeted adenovirus showed longer persistence in the blood circulation with area under plasma concentration-time curve (AUC) values increasing 12-fold compared to unmodified virus. Anti-E-selectin antibody-PEG-adenovirus selectively homed to inflamed skin in mice with a delayed-type hypersensitivity (DTH) inflammation, resulting in local expression of the reporter transgene luciferase. This is the first study showing the benefits of PEGylation on adenovirus behavior upon systemic administration. The approach described here can form the basis for further development of adenoviral gene therapy vectors with improved pharmacokinetics and increased efficiency and specificity of therapeutic gene transfer into endothelial cells in disease. 433 OVERVIEW SUMMARY One of the major limiting factors for gene therapy is the lack of efficiency of gene transfer to desired disease-associated cell types. We have used a novel method for enhancing gene delivery to vascular endothelial cells by coupling cell selective homing ligands (i.e., ␣v integrin-specific RGD peptide and E-selectin-specific antibody) to PEGylated adenovirus. In vitro studies showed that the resulting adenoviral vectors were devoid of transgene transfer into coxsackie-adenovirus receptor (CAR)-positive cells, while specific transgene transfer to activated endothelial cells was achieved. Furthermore, anti-E-selectin antibody-modified PEGylated adenovirus exhibited improved behavior in vivo after systemic administration compared to its unmodified counterpart. It demonstrated an improved blood circulation time, and selectively homed to activated endothelium in skin in mice with a delayed-type hypersensitivity (DTH) skin inflammation, resulting in local expression of the reporter transgene luciferase.
Efficient adenoviral gene transfer to kidney cortical vasculature utilizing a fiber modified vector
The Journal of Gene Medicine, 1999
Background The a v integrin is present in vascular endothelium, including that of the kidney. It is also upregulated in the presence of in¯ammatory cytokines and in some neoplasms. In an effort to transduce vascular endothelial cells, we compare in vitro and in vivo adenoviral gene transfer of a vector with a high af®nity peptide ligand to the a v integrin incorporated into the ®ber coat protein AdZ.F(RGD) to an unmodi®ed vector, AdZ.
Gene Therapy and …, 2004
Adenoviruses type 5 have been successfully exploited as gene transfer vectors and numerous vectorological improvements have contributed to increasing efficiency and specificity of adenoviral gene therapy. Despite these improvements, inefficient gene transfer still is an important limitation and is, at least in part, due to the low expression of the primary receptor (CAR) on target cells. Combining two different fiber types (the fiber of Ad5 for CAR-dependent uptake and the fiber of Ad3 for CAR-independent uptake) on an Ad5-based capsid would increase the options for improvement of specificity and efficiency. In this study, we present an approach to engineer fibermosaic adenoviruses by cloning the fiber of Ad3 into the Ad5 genome under the control of the Major Late Promoter using native splicing signals. Such fiber-mosaic viruses were efficiently rescued using conventional 293 cells and demonstrated good infection profiles. Pre-incubation with recombinant fiber knob (either derived from Ad5 or Ad3) indicated different mechanisms of entry for the fiber-mosaic viruses. The introduction of an additional entry pathway can be further exploited to overcome low infection efficiency due to low CAR expression. In addition, the technology will be of value in increasing the specificity of adenoviral gene therapy since this approach allows the incorporation of two different retargeting ligands per capsid. Such infectivity enhancement will also prove powerful in the context of replicative agents.
Virology, 2004
Many clinically relevant tissues are refractory to Ad5 transduction because of negligible levels of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR). Thus, development of Ad vectors that display CAR-independent tropism could lead directly to therapeutic gain. The Toronto strain of canine adenovirus type 2 (CAV2) exhibits native tropism that is augmented by, but not fully dependent upon, CAR for cellular transduction. We hypothesized that an Ad5 vector containing the nonhuman CAV2 knob would provide expanded tropism and constructed Ad5Luc1-CK, an E1-deleted Ad5 vector encoding the fiber knob domain from CAV2. Ad5Luc1-CK gene delivery to CARdeficient cells was augmented up to 30-fold versus the Ad5 control vector, and correlated with increased cell surface binding. Further, we confirmed the importance of cellular integrins to Ad5Luc1-CK transduction. Herein, we present the rationale, design, purification, and characterization of a novel tropism modified, infectivity-enhanced Ad vector.
Journal of Gene Medicine (2004) 6(3): 309-16., 2004
Background: Modification of the fiber proteins in replication-deficient adenoviral (Ad) vectors through incorporation of specific receptor-binding motifs may represent a strategy to enhance their tissue targeting capabilities.Modification of the fiber proteins in replication-deficient adenoviral (Ad) vectors through incorporation of specific receptor-binding motifs may represent a strategy to enhance their tissue targeting capabilities.Methods: In this study, we compared an unmodified Ad (GV10) with two mutated vectors obtained by insertion of specific target sequences that redirect binding, either toward αV integrin (RGD) or heparan sulfate (UTV) cellular receptors, for reporter gene expression spatial distribution in the rabbit skeletal muscle. In a first series of experiments, injection volume was kept constant and activity of a lacZ transgene was evaluated 48 h after injection of the Ad vectors at different doses. In separate experiments, the effects of different volumes of injection at a constant dose of Ad vector were monitored.In this study, we compared an unmodified Ad (GV10) with two mutated vectors obtained by insertion of specific target sequences that redirect binding, either toward αV integrin (RGD) or heparan sulfate (UTV) cellular receptors, for reporter gene expression spatial distribution in the rabbit skeletal muscle. In a first series of experiments, injection volume was kept constant and activity of a lacZ transgene was evaluated 48 h after injection of the Ad vectors at different doses. In separate experiments, the effects of different volumes of injection at a constant dose of Ad vector were monitored.ResultsAll vectors evaluated showed a significant increase in the number of lacZ-positive muscle segments, with increasing vector dose. However, in muscles treated with the UTV vector, fewer muscle fibers were β-gal-positive than in GV10 or RGD vector treated animals. In fact, total β-gal activity increased in a dose-dependent fashion in the GV10- and RGD-treated muscles, but not in the UTV-treated ones. Remarkably, in samples from UTV-treated animals, a volume-dependent enhancement of transgene expression was observed during experiments performed at the same dose and different injection volumes.All vectors evaluated showed a significant increase in the number of lacZ-positive muscle segments, with increasing vector dose. However, in muscles treated with the UTV vector, fewer muscle fibers were β-gal-positive than in GV10 or RGD vector treated animals. In fact, total β-gal activity increased in a dose-dependent fashion in the GV10- and RGD-treated muscles, but not in the UTV-treated ones. Remarkably, in samples from UTV-treated animals, a volume-dependent enhancement of transgene expression was observed during experiments performed at the same dose and different injection volumes .Conclusions: The results of the present study demonstrate that altering Ad affinity for cellular receptors modulates the level and distribution of transgene activity, conferring characteristics that may allow for treatment customization. Copyright © 2004 John Wiley & Sons, Ltd.The results of the present study demonstrate that altering Ad affinity for cellular receptors modulates the level and distribution of transgene activity, conferring characteristics that may allow for treatment customization. Copyright © 2004 John Wiley & Sons, Ltd.