scholarly journals Targeting Adenoviral Vectors by Using the Extracellular Domain of the Coxsackie-Adenovirus Receptor: Improved Potency via Trimerization

2002 ◽  
Vol 76 (4) ◽  
pp. 1892-1903 ◽  
Author(s):  
Jin Kim ◽  
Theodore Smith ◽  
Neeraja Idamakanti ◽  
Kathy Mulgrew ◽  
Michele Kaloss ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Extracellular Domain ◽  
Adenoviral Vectors ◽  
Target Cells ◽  
Coxsackie Adenovirus Receptor ◽  
Liver Gene ◽  
Peptide Targeting ◽  
Adenovirus Receptor

ABSTRACT Adenovirus binds to mammalian cells via interaction of fiber with the coxsackie-adenovirus receptor (CAR). Redirecting adenoviral vectors to enter target cells via new receptors has the advantage of increasing the efficiency of gene delivery and reducing nonspecific transduction of untargeted tissues. In an attempt to reach this goal, we have produced bifunctional molecules with soluble CAR (sCAR), which is the extracellular domain of CAR fused to peptide-targeting ligands. Two peptide-targeting ligands have been evaluated: a cyclic RGD peptide (cRGD) and the receptor-binding domain of apolipoprotein E (ApoE). Human diploid fibroblasts (HDF) are poorly transduced by adenovirus due to a lack of CAR on the surface. Addition of the sCAR-cRGD or sCAR-ApoE targeting protein to adenovirus redirected binding to the appropriate receptor on HDF. However, a large excess of the monomeric protein was needed for maximal transduction, indicating a suboptimal interaction. To improve interaction of sCAR with the fiber knob, an isoleucine GCN4 trimerization domain was introduced, and trimerization was verified by cross-linking analysis. Trimerized sCAR proteins were significantly better at interacting with fiber and inhibiting binding to HeLa cells. Trimeric sCAR proteins containing cRGD and ApoE were more efficient at transducing HDF in vitro than the monomeric proteins. In addition, the trimerized sCAR protein without targeting ligands efficiently blocked liver gene transfer in normal C57BL/6 mice. However, addition of either ligand failed to retarget the liver in vivo. One explanation may be the large complex size, which serves to decrease the bioavailability of the trimeric sCAR-adenovirus complexes. In summary, we have demonstrated that trimerization of sCAR proteins can significantly improve the potency of this targeting approach in altering vector tropism in vitro and allow the efficient blocking of liver gene transfer in vivo.

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 Role of the putative heparan sulfate glycosaminoglycan-binding site of the adenovirus type 5 fiber shaft on liver detargeting and knob-mediated retargeting

2006 ◽  
Vol 87 (9) ◽  
pp. 2487-2495 ◽  
Author(s):  
Neus Bayo-Puxan ◽  
Manel Cascallo ◽  
Alena Gros ◽  
Meritxell Huch ◽  
Cristina Fillat ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Adenovirus Type ◽  
Adenoviral Vectors ◽  
Coxsackievirus And Adenovirus Receptor ◽  
Protein Ix ◽  
Adenovirus Receptor ◽  
Adenovirus Type 5

Liver tropism hampers systemic administration of adenovirus in gene therapy and virotherapy. In consequence, tumour targeting requires the combination of capsid modifications that abrogate liver transduction and redirect adenoviral vectors to tumour cells. Coxsackievirus and adenovirus receptor (CAR), integrins and heparan sulfate glycosaminoglycans (HSG) are receptors involved in adenovirus type 5 (Ad5) entry into cells. The in vitro and in vivo properties of Ad5 vectors unable to bind CAR, integrins and HSG with and without Arg–Gly–Asp (RGD) inserted at the HI loop of the fiber were studied. As was previously observed with CAR-ablated vectors, CAR and integrin double binding-ablated vectors transduced hepatocytes less efficiently in vitro but not in vivo. On the contrary, the role of HSG on Ad5 infectivity was evident in vitro only when CAR binding was abrogated, but the shaft mutation that ablated HSG binding on the background of a normal capsid was sufficient to abrogate liver transduction in vivo. The insertion of amino acids RGD at the HI loop in a shaft-mutated fiber only partially rescued integrin-mediated infectivity. These results indicate that the shaft mutation precluded HSG binding and affected the structure of the fiber. The insertion of ligands at the hexon or protein IX may be required to benefit from the fiber shaft mutation-detargeting properties.

Limiting Carryover Events Resulting from Cell Surface Retention of VSV-G Pseudotyped HIV-Lentivector Particles on Hematopoietic Targets

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3735-3735
Author(s):  
Lee O’Neill ◽  
Yung-Wei Pan ◽  
Amy M. Skinner ◽  
Peter Kurre
Keyword(s):  
Gene Transfer ◽  
Cell Surface ◽  
Ex Vivo ◽  
Target Cells ◽  
Vector Particle ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Vector Particles

Abstract Preclinical evidence and clinical trials speak to the therapeutic potential of retrovirus vectors for the heritable genetic modification of cells. Careful evaluation of the antecedent risks is critical to move these applications forward. Others previously demonstrated the persistence of intact vector particles on the surface of target cells. Inadvertent particle transfer after in vivo applications could lead to the transduction of bystander tissues, or provoke immunological responses. We recently demonstrated prolonged adherence of VSV-G pseudotyped, HIV-1 derived lentivirus particles after ex vivo transduction culture of murine hematopoietic target cells (1°) with subsequent transduction of secondary (2°) targets in vitro and in vivo. Extended particle adherence is independent of Env pseudotype and routine wash procedures (Pan et al., J Virol. Jan 2007). We hypothesized that unwanted carryover could be minimized by disrupting the vector particle attachment to 2° cells while maintaining uptake to 1° targets. Initial studies indicated that the transduction of 1° targets at 4°C (to prevent uptake) for up to 6 hours followed by serial PBS washes and subsequent direct co-culture with fibroblasts resulted in undiminished 2° gene transfer compared to transduction at 37°C. Conversely, post-transduction exposure to escalating concentrations of citric acid resulted in a systematic decrease in both 1° and 2° gene transfer rates. This is consistent with separable mechanisms for pH sensitive VSV-G mediated uptake of particles in 1° targets and the receptor independent attachment responsible for carryover and 2° transduction, respectively. Glycosaminoglycans, including heparin, quantitatively bind to pseudotyped vector particles. We found that exposure of particles to heparin effectively abrogated subsequent transduction of cells by disrupting attachment. Remarkably, serial heparin washes at the conclusion of transduction had only minimal effects on gene transfer to 1° targets, but resulted in a two-log reduction in 2° gene transfer. Increases in the concentration of protamine sulfate (a polycation) during transduction partly reversed the effect of heparin (a polyanion), demonstrating the residual impact of electrostatic interactions on attachment of retrovirus particles from the 1° cell. In further studies we showed that trypsin washes following vector exposure incompletely cleaved 1° cell surface bound particles while pronase effectively degraded cell surface bound particles in a dose dependent manner, abrogating carryover. Because pronase at high concentrations also compromised cell surface epitope integrity we studied the expression of chemokine receptor (CXCR) 4, both a critical mediator of progenitor cell homing to the bone marrow and a representative protease-sensitive surface molecule. These experiments revealed a dose dependent degradation of CXCR4 on the cell surface of 1° target cells and rapid regeneration within three hours, critical for applications involving the injection of ex vivo modified hematopoietic cells. In conclusion, our results demonstrate that select wash procedures can disrupt the ability of virus particles to bind secondary targets, degrade residual surface bound particles and reduce gene transfer to inadvertent 2° targets in vitro by up to 99%. These studies are important first steps in understanding and limiting inadvertent carryover in the context of gene therapy while maximizing target cell transduction.

scholarly journals Reduction of Natural Adenovirus Tropism to the Liver by both Ablation of Fiber-Coxsackievirus and Adenovirus Receptor Interaction and Use of Replaceable Short Fiber

2003 ◽  
Vol 77 (4) ◽  
pp. 2512-2521 ◽  
Author(s):  
Takafumi Nakamura ◽  
Kenzo Sato ◽  
Hirofumi Hamada
Keyword(s):  
Gene Transfer ◽  
Adenovirus Vector ◽  
Short Fiber ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Shaft Length ◽  
Adenovirus Serotype ◽  
Coxsackievirus And Adenovirus Receptor ◽  
Adenovirus Receptor

ABSTRACT The initial recognition and binding of adenovirus vector to the host cell surface is mediated by interaction between the adenovirus fiber knob protein and its receptor, the coxsackievirus and adenovirus receptor (CAR). This natural tropism of adenovirus vector needs to be ablated in order to achieve targeted gene transfer. To this end, we noted that adenovirus serotype 40 (Ad40) contains two distinct long and short fibers; the short fiber is unable to recognize CAR, while the long fiber binds CAR. We generated adenovirus serotype 5-based mutants with chimeric Ad40-derived fibers, which were composed of either long or short shafts together with CAR binding or nonbinding knobs. The capacity of these adenovirus mutants for in vitro and in vivo gene transfer to liver cells was examined. In the case of primary human hepatocytes displaying a high expression level of CAR and αv integrin, both CAR binding ability and fiber shaft length played important roles in efficient transduction. Most significantly, the high transduction efficiency observed in the liver and spleen following intravenous administration of adenovirus vector was dramatically reduced by both ablation of fiber-CAR interaction and the use of replaceable short fiber. In other tissues displaying a low level of transduction, no significant differences in transduction efficiency were observed among adenovirus vector mutants. Furthermore, incorporation of a 7-lysine-residue motif at the C-terminal end of CAR-nonbinding short fiber efficiently achieved transduction of target cells via the heparan-containing receptor. Our results demonstrated that the natural tropism of adenovirus in vivo is influenced not only by fiber-CAR interaction but also by fiber shaft length. Furthermore, our strategy may be useful for retargeting adenovirus to particular tumors and tissue types with specific receptors.

scholarly journals Advantages of Using Paclitaxel in Combination with Oncolytic Adenovirus Utilizing RNA Destabilization Mechanism

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1210 ◽  
Author(s):  
Elora Hossain ◽  
Umma Habiba ◽  
Aya Yanagawa-Matsuda ◽  
Arefin Alam ◽  
Ishraque Ahmed ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cytolytic Activity ◽  
Oncolytic Viruses ◽  
Coxsackie Adenovirus Receptor ◽  
Novel Approach ◽  
E1a Gene ◽  
Adenovirus Receptor ◽  
Conditionally Replicative Adenovirus

Oncolytic virotherapy is a novel approach to cancer therapy. Ad-fosARE is a conditionally replicative adenovirus engineered by inserting AU-rich elements (ARE) in the 3’-untranslated region of the E1A gene. In this study, we examined the oncolytic activity of Ad-fosARE and used it in a synergistic combination with the chemotherapeutic agent paclitaxel (PTX) for treating cancer cells. The expression of E1A was high in cancer cells due to stabilized E1A-ARE mRNA. As a result, the efficiency of its replication and cytolytic activity in cancer cells was higher than in normal cells. PTX treatment increased the cytoplasmic HuR relocalization in cancer cells, enhanced viral replication through elevated E1A expression, and upregulated CAR (Coxsackie-adenovirus receptor) required for viral uptake. Furthermore, PTX altered the instability of microtubules by acetylation and detyrosination, which is essential for viral internalization and trafficking to the nucleus. These results indicate that PTX can provide multiple advantages to the efficacy of Ad-fosARE both in vitro and in vivo, and provides a basis for designing novel clinical trials. Thus, this virus has a lot of benefits that are not found in other oncolytic viruses. The virus also has the potential for treating PXT-resistant cancers.

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