Efficient gene transfer by fiber-mutant adenoviral vectors containing RGD peptide

One of the hurdles to adenovirus (Ad)-mediated gene transfer is that Ad vectors mediate inefficient gene transfer into cells lacking in the primary receptors, Coxsackievirus and adenovirus receptor (CAR). We previously developed a fiber-mutant Ad vector containing the Arg-Gly-Asp (RGD)-containing peptide motif on the HI loop of the fiber knob, and showed that the mutant vector had enhanced gene transfer activity to human glioma cells, which showed little CAR expression, compared to the vector containing wild type fiber. In this study, the feasibility of the Ad vector containing RGD peptide on the fiber knob was examined in a wide variety of cell types: CAR-positive or -negative human tumor cells, mouse cells, and leukemia cells. The mutant vector infected the cells, which lacked CAR expression but showed αv integrin expression, about 10–1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (αvβ3 and αvβ5)-dependent, CAR-independent cell entry pathway. The results of this study indicate that Ad vector containing RGD peptide on the fiber knob could be of great utility for gene therapy and gene transfer experiments.     

Reduction of natural adenovirus tropism to mouse liver by fiber-shaft exchange in combination with both CAR- and alphav integrin-binding ablation

The primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, αv integrins, are the tropism determinants of adenovirus (Ad) type 5. Inhibition of the interaction of both the fiber with CAR and the penton base with the αv integrin appears to be crucial to the development of targeted Ad vectors, which specifically transduce a given cell population. In this study, we developed Ad vectors with ablation of both CAR and αv integrin binding by mutating the fiber knob and the RGD motif of the penton base. We also replaced the fiber shaft domain with that derived from Ad type 35. High transduction efficiency in the mouse liver was suppressed approximately 130- to 270-fold by intravenous administration of the double-mutant Ad vectors, which mutated two domains each of the fiber knob and shaft and the RGD motif of the penton base compared with those of conventional Ad vectors (type 5). Most significantly, the triple-mutant Ad vector containing the fiber knob with ablation of CAR binding ability, the fiber shaft of Ad type 35, and the penton base with a deletion of the RGD motif mediated a >30,000-fold lower level of mouse liver transduction than the conventional Ad vectors. This triple-mutant Ad vector also mediated reduced transduction in other organs (the spleen, kidney, heart, and lung). Viral DNA analysis showed that systemically delivered triple-mutant Ad vector was primarily taken up by liver nonparenchymal cells and that most viral DNAs were easily degraded, resulting in little gene expression in the liver. These results suggest that the fiber knob, fiber shaft, and RGD motif of the penton base each plays an important role in Ad vector-mediated transduction to the mouse liver and that the triple-mutant Ad vector exhibits little tropism to any organs and appears to be a fundamental vector for targeted Ad vectors.     

Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad with a transfer vector containing both the E4 region and the modified fiber gene. AdZ.F(RGD) increased gene delivery to endothelial and smooth muscle cells expressing alpha(v) integrins. Likewise, AdZ.F(pK7) increased transduction 5- to 500-fold in multiple cell types lacking high levels of Ad fiber receptor, including macrophage, endothelial, smooth muscle, fibroblast, and T cells. In addition, AdZ.F(pK7) significantly increased gene transfer in vivo to vascular smooth muscle cells of the porcine iliac artery following balloon angioplasty. These vectors may therefore be useful in gene therapy for vascular restenosis or for targeting endothelial cells in tumors. Although binding to the fiber receptor still occurs with these vectors, they demonstrate the feasibility of tissue-specific receptor targeting in cells which express low levels of Ad fiber receptor.     

Canine adenovirus type 2 attachment and internalization: coxsackievirus-adenovirus receptor, alternative receptors, and an RGD-independent pathway

The best-characterized receptors for adenoviruses (Ads) are the coxsackievirus-Ad receptor (CAR) and integrins alpha(v)beta(5) and alpha(v)beta(3), which facilitate entry. The alpha(v) integrins recognize an Arg-Gly-Asp (RGD) motif found in some extracellular matrix proteins and in the penton base in most human Ads. Using a canine adenovirus type 2 (CAV-2) vector, we found that CHO cells that express CAR but not wild-type CHO cells are susceptible to CAV-2 transduction. Cells expressing alpha(M)beta(2) integrins or major histocompatibility complex class I (MHC-I) molecules but which do not express CAR were not transduced. Binding assays showed that CAV-2 attaches to a recombinant soluble form of CAR and that Ad type 5 (Ad5) fiber, penton base, and an anti-CAR antibody partially blocked attachment. Using fluorescently labeled CAV-2 particles, we found that in some cells nonpermissive for transduction, inhibition was at the point of internalization and not attachment. The transduction efficiency of CAV-2, which lacks an RGD motif, surprisingly mimicked that of Ad5 when tested in cells selectively expressing alpha(v)beta(5) and alpha(v)beta(3) integrins. Our results demonstrate that CAV-2 transduction is augmented by CAR and possibly by alpha(v)beta(5), though transduction can be CAR and alpha(v)beta(3/5) independent but is alpha(M)beta(2), MHC-I, and RGD independent, demonstrating a transduction mechanism which is distinct from that of Ad2/5.     

Efficient gene transfer into human CD34(+) cells by a retargeted adenovirus vector

Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and alpha(v) integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34(+) cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34(+) cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an alpha(v) integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34(+) cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34(+) cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34(+) cells expressing alpha(v) integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34(+) cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34(+) cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34(+) c-Kit(+) cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34(+) c-Kit(+) cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells.     

Influence of fiber detargeting on adenovirus-mediated innate and adaptive immune activation

The major adenovirus (Ad) capsid proteins hexon, penton, and fiber influence the efficiency and tropism of gene transduction by Ad vectors. Fiber is the high-affinity receptor binding protein that serves to mediate cell attachment in vitro when using coxsackie-adenovirus receptor (CAR)-containing cell lines. This contrasts with transduction efficiency in macrophages or dendritic cells that lack high concentrations of CAR. To determine how fiber influences gene transduction and immune activation in a murine model, we have characterized Ad type 5 (Ad5) vectors with two classes of chimeric fiber, CAR binding and non-CAR binding. In a systemic infection, Ad5 fiber contributes to DNA localization and vector transduction in hepatic tissue. However, the majority of vector localization is due to Ad5 fiber-specific functions distinct from CAR binding. CAR-directed transduction occurs but at a modest level. In contrast to CAR binding vectors, the F7 and F7F41S non-CAR-binding vectors demonstrate a 2-log decrease in hepatic transduction, with a 10-fold decrease in the amount of vector DNA localizing to the hepatic tissue. To characterize the innate response to early infection using fiber chimeric vectors, intrahepatic cytokine and chemokine mRNAs were quantified 5 hours postinfection. Tumor necrosis factor alpha mRNA levels resulting from Ad5 fiber infections were elevated compared to viruses expressing serotype 7 or 41 fiber. Levels of chemokine mRNA (gamma interferon-inducible protein 10, T-cell activation gene 3, and macrophage inflammatory protein 1beta) were 10- to 20-fold higher with CAR binding vectors (Ad5 and F41T) than with non-CAR-binding vectors (F7 and F7F41S). In spite of quantitative differences in vector localization and innate activation, fiber pseudotyping did not significantly change the outcome of anti-Ad adaptive immunity. All vectors were cleared with the same kinetics as wild-type Ad5 vectors, and each induced neutralizing antibody. Although non-CAR-binding vectors were impaired in transduction by nearly 2 orders of magnitude, the level of antitransgene immunity was the same for each of the vectors. Using primary bone marrow-derived macrophages and dendritic cells, we demonstrate that transduction, induction of cytokine/chemokine, and phenotypic maturation of these antigen-presenting cells are independent of fiber content. Our data support a model where fiber-mediated hepatic localization enhances innate responses to virus infection but minimally impacts on adaptive immunity.     

Novel fiber-dependent entry mechanism for adenovirus serotype 5 in lacrimal acini

The established mechanism for infection of most cells with adenovirus serotype 5 (Ad5) involves fiber capsid protein binding to coxsackievirus-adenovirus receptor (CAR) at the cell surface, followed by penton base capsid protein binding to alpha(v) integrins, which triggers clathrin-mediated endocytosis of the virus. Here we determined the identity of the capsid proteins responsible for mediating Ad5 entry into the acinar epithelial cells of the lacrimal gland. Ad5 transduction of primary rabbit lacrimal acinar cells was inhibited by excess Ad5 fiber or knob (terminal region of the fiber) but not excess penton base. Investigation of the interactions of recombinant Ad5 penton base, fiber, and knob with lacrimal acini revealed that the penton base capsid protein remained surface associated, while the knob domain of the fiber capsid protein was rapidly internalized. Introduction of rabbit CAR-specific small interfering RNA (siRNA) into lacrimal acini under conditions that reduced intracellular CAR mRNA significantly inhibited Ad5 transduction, in contrast to a control (nonspecific) siRNA. Preincubation of Ad5 with excess heparin or pretreatment of acini with a heparinase cocktail each inhibited Ad5 transduction by a separate and apparently additive mechanism. Functional and imaging studies revealed that Ad5, fiber, and knob, but not penton base, stimulated macropinocytosis in acini and that inhibition of macropinocytosis significantly reduced Ad5 transduction of acini. However, inhibition of macropinocytosis did not reduce Ad5 uptake. We propose that internalization of Ad5 into lacrimal acini is through a novel fiber-dependent mechanism that includes CAR and heparan sulfate glycosaminoglycans and that the subsequent intracellular trafficking of Ad5 is enhanced by fiber-induced macropinocytosis.     

Adenovirus type 5 viral particles pseudotyped with mutagenized fiber proteins show diminished infectivity of coxsackie B-adenovirus receptor-bearing cells

A major limitation of adenovirus type 5 (Ad5)-based gene therapy, the inability to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-adenovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domain have been shown to alter the tropism of the virus. We have developed a novel system to rapidly evaluate the function of modified fiber proteins in their most relevant context, the adenoviral capsid. This transient transfection/infection system combines transfection of cells with plasmids that express high levels of the modified fiber protein and infection with Ad5.beta gal.Delta F, an E1-, E3-, and fiber-deleted adenoviral vector encoding beta-galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were incorporated via mutagenesis into the fiber expression plasmid, and the resulting fiber proteins were subsequently incorporated onto adenoviral particles. Mutations located in the fiber knob AB and CD loops demonstrated the greatest reduction in fiber-mediated gene transfer in HeLa cells. We also observed effects on transduction efficiency with mutations in the FG loop, indicating that the binding site may extend to the adjacent monomer in the fiber trimer and in the HI loop. These studies support the concept that modification of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneously with novel ligands for the development of a systemically administered, targeted adenoviral vector.     

Ocular Localization and Transduction by Adenoviral Vectors Are Serotype-Dependent and Can Be Modified by Inclusion of RGD Fiber Modifications

Purpose

To evaluate localization and transgene expression from adenoviral vector of serotypes 5, 35, and 28, ± an RGD motif in the fiber following intravitreal or subretinal administration.

Methods

Ocular transduction by adenoviral vector serotypes ± RGD was studied in the eyes of mice receiving an intravitreous or subretinal injection. Each serotype expressed a CMV-GFP expression cassette and histological sections of eyes were examined. Transgene expression levels were examined using luciferase (Luc) regulated by the CMV promoter.

Results

GFP localization studies revealed that serotypes 5 and 28 given intravitreously transduced corneal endothelial, trabecular, and iris cells. Intravitreous delivery of the unmodified Ad35 serotype transduced only trabecular meshwork cells, but, the modification of the RGD motif into the fiber of the Ad35 viral vector base expanded transduction to corneal endothelial and iris cells. Incorporation of the RGD motif into the fiber knob with deletion of RGD from the penton base did not affect the transduction ability of the Ad5 vector base. Subretinal studies showed that RGD in the Ad5 knob shifted transduction from RPE cells to photoreceptor cells. Using a CMV-Luc expression cassette, intravitreous delivery of all the tested vectors, such as Ad5-, Ad35- and Ad28- resulted in an initial rapid induction of luciferase activity that thereafter declined. Subretinal administration of vectors showed a marked difference in transgene activity. Ad35-Luc gene expression peaked at 7 days and remained elevated for 6 months. Ad28-Luc expression was high after 1 day and remained sustained for one month.

Conclusions

Different adenoviral vector serotypes ± modifications transduce different cells within the eye. Transgene expression can be brief or extended and is serotype and delivery route dependent. Thus, adenoviral vectors provide a versatile platform for the delivery of therapeutic agents for ocular diseases.     

An Adenovirus Vector with Genetically Modified Fibers Demonstrates Expanded Tropism via Utilization of a Coxsackievirus and Adenovirus Receptor-Independent Cell Entry Mechanism

Recombinant adenoviruses (Ad) have become the vector system of choice for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to cells expressing marginal levels of the coxsackievirus and adenovirus receptor (CAR). In order to achieve CAR-independent gene transfer by Ad vectors in clinically important contexts, we proposed modification of viral tropism via genetic alterations to the viral fiber protein. We have shown that incorporation of an Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the fiber knob domain results in the ability of the virus to utilize an alternative receptor during the cell entry process. We have also demonstrated that due to its expanded tissue tropism, this novel vector is capable of efficient transduction of primary tumor cells. An increase in gene transfer to ovarian cancer cells of 2 to 3 orders of magnitude was demonstrated by the vector, suggesting that recombinant Ad containing fibers with an incorporated RGD peptide may be of great utility for treatment of neoplasms characterized by deficiency of the primary Ad type 5 receptor.     

Fiber and penton base capsid modifications yield diminished adenovirus type 5 transduction and proinflammatory gene expression with retention of antigen-specific humoral immunity

Fiber and penton base capsid proteins of adenovirus type 5 (Ad5) mediate a well-characterized two-step entry pathway in permissive tissue culture cell lines. Fiber binds with high affinity to the cell surface coxsackievirus-and-adenovirus receptor (CAR), and penton base facilitates viral internalization by binding alphav integrins through an RGD motif. In vivo, the entry pathway is complicated by interactions of capsid proteins with additional cell surface molecules and blood factors. When administered systemically in mice, adenovirus vectors (Adv) localize primarily to hepatic tissue, resulting in efficient gene transduction and potent activation of the host antiviral immune response. The goal of the present study was to detarget Adv uptake through fiber and penton base capsid protein manipulations and determine how detargeted vectors influence transduction efficiency, inflammatory activation, and activation of the adaptive arm of the immune system. By manipulating fiber and the penton base, we have generated highly detargeted vectors (up to 1,200-fold reduction in transgene expression in vivo) with reduced macrophage stimulatory activity in vitro and in vivo. In spite of the diminished transduction and macrophage activation, the detargeted vectors induce strong neutralizing immunity as well as efficient antitransgene antibody. Three of the modified vectors produce antitransgene humoral immunity at levels that exceed or are equal to that seen with an unmodified Ad5-based vector. The fiber-pseudotyped and penton base constructs with RGD deleted have attributes that could be important enhancements in a number of vaccine applications.     

Dependence of adenovirus infectivity on length of the fiber shaft domain

One of the objectives in adenovirus (Ad) vector development is to target gene delivery to specific cell types. Major attention has been given to modification of the Ad fiber knob, which is thought to determine virus tropism. However, among the human Ad serotypes with different tissue tropisms, not only the knob but also the length of the fiber shaft domain varies significantly. In this study we attempted to delineate the role of fiber length in coxsackievirus-adenovirus receptor (CAR)- and non-CAR-mediated infection. A series of Ad serotype 5 (Ad5) capsid-based vectors containing long or short fibers with knob domains derived from Ad5, Ad9, or Ad35 was constructed and tested in adsorption, internalization, and transduction studies. For Ad5 or Ad9 knob-possessing vectors, a long-shafted fiber was critical for efficient adsorption/internalization and transduction of CAR/alphav integrin-expressing cells. Ad5 capids containing short CAR-recognizing fibers were affected in cell adsorption and infection. In contrast, for the chimeric vectors possessing Ad35 knobs, which enter cells by a CAR/alphav integrin-independent pathway, fiber shaft length had no significant influence on binding or infectibility on tested cells. The weak attachment of short-shafted Ad5 or Ad9 knob-possessing vectors seems to be causally associated with a charge-dependent repulsion between Ad5 capsid and acidic cell surface proteins. The differences between short- and long-shafted vectors in attachment or infection were abrogated by preincubation of cells with polycations. This study demonstrates that the fiber-CAR interaction is not the sole determinant for tropism of Ad vectors containing chimeric fibers. CAR- and alphav integrin-mediated infections are influenced by other factors, including the length of the fiber shaft.     

Protective immunity to pseudomonas aeruginosa induced with a capsid-modified adenovirus expressing P. aeruginosa OprF

This study focuses on the development of a new clinical vaccine candidate (AdOprF.RGD.Epi8) against Pseudomonas aeruginosa using an E1⁻ E3⁻ adenovirus (Ad) vector expressing OprF (AdOprF.RGD.Epi8) and modifications of the Ad genome providing two capsid changes: (i) modification of the Ad hexon gene to incorporate an immune-dominant OprF epitope (Epi8) into loop 1 of the hexon, enabling repeat administration to boost the anti-OprF immune response, and (ii) modification of the fiber gene to incorporate an integrin-binding RGD sequence to enhance gene delivery to antigen-presenting cells. Western analysis confirmed that AdOprF.RGD.Epi8 expresses OprF, contains Epi8 in the hexon protein, and enhances gene transfer to dendritic cells compared to AdOprF, a comparable Ad vector expressing OprF with an unmodified capsid. Intramuscular immunization of C57BL/6 mice with AdOprF.RGD.Epi8 resulted in the generation of anti-OprF antibodies at comparable levels to those induced following immunization with AdOprF, but immunization with AdOprF.RGD.Epi8 was associated with increased CD4 and CD8 gamma interferon T-cell responses against OprF as well as increased survival against lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeat administration of AdOprF.RGD.Epi8 resulted in boosting of the humoral anti-OprF response as well as increased protection, whereas no boosting could be achieved with repeat administration of AdOprF. This suggests that the capsid-modified AdOprF.RGD.Epi8 vector is a more effective immunogen compared to a comparable wild-type Ad capsid, making it a good candidate for an anti-P. aeruginosa vaccine.     

Reovirus sigma1 fiber incorporated into adenovirus serotype 5 enhances infectivity via a CAR-independent pathway

Adenovirus serotype 5 (Ad5) has been used for gene therapy with limited success because of insufficient infectivity in cells with low expression of the primary receptor, the coxsackie and adenovirus receptor (CAR). To enhance infectivity in tissues with low CAR expression, tropism expansion is required via non-CAR pathways. Serotype 3 Dearing reovirus utilizes a fiber-like sigma1 protein to infect cells expressing sialic acid and junction adhesion molecule 1 (JAM1). We hypothesized that replacement of the Ad5 fiber with sigma1 would result in an Ad5 vector with CAR-independent tropism. We therefore constructed a fiber mosaic Ad5 vector, designated as Ad5-sigma1, encoding two fibers: the sigma1 and the wild-type Ad5 fiber. Functionally, Ad5-sigma1 utilized CAR, sialic acid, and JAM1 for cell transduction and achieved maximum infectivity enhancement in cells with or without CAR. Thus, we have developed a new type of Ad5 vector with expanded tropism, possessing fibers from Ad5 and reovirus, that exhibits enhanced infectivity via CAR-independent pathway(s).     

Identification of HI-like loop in CELO adenovirus fiber for incorporation of receptor binding motifs

Vectors based on the chicken embryo lethal orphan (CELO) avian adenovirus (Ad) have two attractive properties for gene transfer applications: resistance to preformed immune responses to human Ads and the ability to grow in chicken embryos, allowing low-cost production of recombinant viruses. However, a major limitation of this technology is that CELO vectors demonstrate decreased efficiency of gene transfer into cells expressing low levels of the coxsackie-Ad receptor (CAR). In order to improve the efficacy of gene transfer into CAR-deficient cells, we modified viral tropism via genetic alteration of the CELO fiber 1 protein. The alphav integrin-binding motif (RGD) was incorporated at two different sites of the fiber 1 knob domain, within an HI-like loop that we identified and at the C terminus. Recombinant fiber-modified CELO viruses were constructed containing secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein genes as reporter genes. Our data show that insertion of the RGD motif within the HI-like loop of the fiber resulted in significant enhancement of gene transfer into CAR-negative and CAR-deficient cells. In contrast, CELO vectors containing the RGD motif at the fiber 1 C terminus showed reduced transduction of all cell lines. CELO viruses modified with RGD at the HI-like loop transduced the SEAP reporter gene into rabbit mammary gland cells in vivo with an efficiency significantly greater than that of unmodified CELO vector and similar to that of Ad type 5 vector. These results illustrate the potential for efficient CELO-mediated gene transfer into a broad range of cell types through modification of the identified HI-like loop of the fiber 1 protein.     

Genetic Retargeting of Adenovirus: Novel Strategy Employing “Deknobbing” of the Fiber

For efficient and versatile use of adenovirus (Ad) as an in vivo gene therapy vector, modulation of the viral tropism is highly desirable. In this study, a novel method to genetically alter the Ad fiber tropism is described. The knob and the last 15 shaft repeats of the fiber gene were deleted and replaced with an external trimerization motif and a new cell-binding ligand, in this case the integrin-binding motif RGD. The corresponding recombinant fiber retained the basic biological functions of the natural fiber, i.e., trimerization, nuclear import, penton formation, and ligand binding. The recombinant fiber bound to integrins but failed to react with antiknob antibody. For virus production, the recombinant fiber gene was rescued into the Ad genome at the exact position of the wild-type (WT) fiber to make use of the native regulation of fiber expression. The recombinant virus Ad5/FibR7-RGD yielded plaques on 293 cells, but the spread through the monolayer was two to three times delayed compared to WT, and the ratio of infectious to physical particles was 20 times lower. Studies on virus tropism showed that Ad5/FibR7-RGD was able to infect cells which did not express the coxsackie-adenovirus receptor (CAR), but did express integrins. Ad5/FibR7-RGD virus infectivity was unchanged in the presence of antiknob antibody, which neutralized the WT virus. Ad5/FibR7-RGD virus showed an expanded tropism, which is useful when gene transfer to cells not expressing CAR is needed. The described method should also make possible the construction of Ad genetically retargeted via ligands other than RGD.     

Subgroup B and F fiber chimeras eliminate normal adenovirus type 5 vector transduction in vitro and in vivo

Altering adenovirus vector (Ad vector) targeting is an important goal for a variety of gene therapy applications and involves eliminating or reducing the normal tropism of a vector and retargeting through a distinct receptor-ligand pathway. The first step of Ad vector infection is high-affinity binding to a target cellular receptor. For the majority of adenoviruses and Ad vectors, the fiber capsid protein serves this purpose, binding to the coxsackievirus and adenovirus receptor (CAR) present on a variety of cell types. In this study we have explored a novel approach to altering Ad type 5 (Ad5) vector targeting based on serotypic differences in fiber function. The subgroup B viruses bind to an unidentified receptor that is distinct from CAR. The subgroup F viruses are the only adenoviruses that express two distinct terminal exons encoding fiber open reading frames. We have constructed chimeric fiber adenoviruses that utilize the tandem fiber arrangement of the subgroup F genome configuration. By taking advantage of serotypic differences in fiber expression, fiber shaft length, and fiber binding efficiency, we have developed a tandem fiber vector that has low binding efficiency for the known fiber binding sites, does not rely on an Ad5-based fiber, and can be grown to high titer using conventional cell lines. Importantly, when characterizing these vectors in vivo, we find the subgroup B system and our optimal tandem fiber system demonstrate reduced liver transduction by over 2 logs compared to an Ad5 fiber vector. These attributes make the tandem fiber vector a useful alternative to conventional strategies for fiber manipulation of adenovirus vectors.     

Conserved Fiber-Penton Base Interaction Revealed by Nearly Atomic Resolution Cryo-Electron Microscopy of the Structure of Adenovirus Provides Insight into Receptor Interaction

Adenovirus (Ad) cell attachment is initiated by the attachment of the fiber protein to a primary receptor (usually CAR or CD46). This event is followed by the engagement of the penton base protein with a secondary receptor (integrin) via its loop region, which contains an Arg-Gly-Asp (RGD) motif, to trigger virus internalization. To understand the well-orchestrated adenovirus cell attachment process that involves the fiber and the penton base, we reconstructed the structure of an Ad5F35 capsid, comprising an adenovirus type 5 (Ad5) capsid pseudotyped with an Ad35 fiber, at a resolution of approximately 4.2 Å. The fiber-penton base interaction in the cryo-electron microscopic (cryo-EM) structure of Ad5F35 is similar to that in the cryo-EM structure of Ad5, indicating that the fiber-penton base interaction of adenovirus is conserved. Our structure also confirms that the C-terminal segment of the fiber tail domain constitutes the bottom trunk of the fiber shaft. Based on the conserved fiber-penton base interaction, we have proposed a model for the interaction of Ad5F35 with its primary and secondary receptors. This model could provide insight for designing adenovirus gene delivery vectors.     

Adenovirus vectors containing chimeric type 5 and type 35 fiber proteins exhibit altered and expanded tropism and increase the size limit of foreign genes

Adenovirus (Ad) fiber proteins are responsible for the initial attachment of the virion to the cell membrane. Most Ad vectors currently in use are based on the Ad type 5 (Ad5), which belong to subgroup C, and use the coxsackievirus and adenovirus receptors (CAR) as the initial receptor. Ad35, which belongs to subgroup B, recognizes unknown receptor(s) other than CAR. In this study, the feasibility of the Ad vector containing Ad5/35 chimeric fiber protein was examined in a wide variety of cell types, such as CAR-positive or -negative human tumor cells, rodent cells, and blood cells (a total of 20 cell types), and in mice in vivo. Transduction data suggested that the Ad vectors containing the Ad5/35 chimeric fiber protein exhibited altered and expanded tropism when compared with the Ad5-based vector. The chimeric vector also allows the packaging of larger foreign DNAs than the conventional Ad5-based vector, which can package approximately 8.1-8.2 kb of foreign DNA. The chimeric vector containing approximately 8.8 kb of foreign DNA was generated without affecting the viral growth rate and titer. These results suggested that inclusion of the Ad35 fiber protein into the Ad5-based vector could lead to an improved efficiency in gene therapy and in gene transfer experiments, especially for the cells lacking in sufficient CAR expression.