Development and Assessment of Human Adenovirus Type 11 as a Gene Transfer Vector

ABSTRACT Adenovirus vectors based on human serotype 5 (Ad5) have successfully been used as gene transfer vectors in many gene therapy-based approaches to treat disease. Despite their widespread application, many potential therapeutic applications are limited by the widespread prevalence of vector-neutralizing antibodies within the human population and the inability of Ad5-based vectors to transduce important therapeutic target cell types. In an attempt to circumvent these problems, we have developed Ad vectors based on human Ad serotype 11 (Ad11), since the prevalence of neutralizing antibodies to Ad11 in humans is low. E1-deleted Ad11 vector genomes were generated by homologous recombination in 293 cells expressing the Ad11-E1B55K protein or by recombination in Escherichia coli. E1-deleted Ad11 genomes did not display transforming activity in rodent cells. Transduction of primary human CD34+ hematopoietic progenitor cells and immature dendritic cells was more efficient with Ad11 vectors than with Ad5 vectors. Thirty minutes after intravenous injection into mice that express one of the Ad11 receptors (CD46), we found, in a pattern and at a level comparable to what is found in humans, Ad11 vector genomes in all analyzed organs, with the highest amounts in liver, lung, kidney, and spleen. Neither Ad11 genomes nor Ad11 vector-mediated transgene expression were, however, detected at 72 h postinfusion. A large number of Ad11 particles were also found to be associated with circulating blood cells. We also discovered differences in in vitro transduction efficiencies and in vivo biodistributions between Ad11 vectors and chimeric Ad5 vectors possessing Ad11 fibers, indicating that Ad11 capsid proteins other than fibers influence viral infectivity and tropism. Overall, our study provides a basis for the application of Ad11 vectors for in vitro and in vivo gene transfer and for gaining an understanding of the factors that determine Ad tropism.

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In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

Viruses ◽

10.3390/v13081483 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1483

Author(s):

Emily A. Bates ◽

John R. Counsell ◽

Sophie Alizert ◽

Alexander T. Baker ◽

Natalie Suff ◽

...

Keyword(s):

Viral Vector ◽

Ex Vivo ◽

Human Adenovirus ◽

Adenovirus Type ◽

Cell Types ◽

In Vivo Evaluation ◽

In Vivo Biodistribution ◽

Adenovirus Type 5

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

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In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

10.20944/preprints202107.0153.v2 ◽

2021 ◽

Author(s):

Emily A. Bates ◽

John R. Counsell ◽

Sophie Alizert ◽

Alexander T. Baker ◽

Natalie Suff ◽

...

Keyword(s):

Viral Vector ◽

Ex Vivo ◽

Human Adenovirus ◽

Adenovirus Type ◽

Cell Types ◽

In Vivo Evaluation ◽

In Vivo Biodistribution ◽

Adenovirus Type 5

The human adenovirus phylogenetic tree is split across seven species (A-G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of preexisting immunity detected across screened populations. However, many aspects of the basic virology of species D, such as their cellular tropism, receptor usage and in vivo biodistribution profile, remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49), a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen whilst avoiding liver interactions, such as intravascular vaccine applications.

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In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

10.20944/preprints202107.0153.v1 ◽

2021 ◽

Author(s):

Emily A. Bates ◽

John R. Counsell ◽

Sophie Alizert ◽

Alexander T. Baker ◽

Natalie Suff ◽

...

Keyword(s):

Viral Vector ◽

Ex Vivo ◽

Human Adenovirus ◽

Adenovirus Type ◽

Cell Types ◽

In Vivo Evaluation ◽

In Vivo Biodistribution ◽

Adenovirus Type 5

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

Journal of Virology ◽

10.1128/jvi.80.3.1487-1496.2006 ◽

2006 ◽

Vol 80 (3) ◽

pp. 1487-1496 ◽

Cited By ~ 26

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.

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Canine Adenovirus Vectors: an Alternative for Adenovirus-Mediated Gene Transfer

Journal of Virology ◽

10.1128/jvi.74.1.505-512.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 505-512 ◽

Cited By ~ 188

Author(s):

Eric J. Kremer ◽

Sylvie Boutin ◽

Miguel Chillon ◽

Olivier Danos

Keyword(s):

Gene Transfer ◽

Neutralizing Antibodies ◽

Viral Vectors ◽

Human Adenovirus ◽

Adenovirus Type ◽

Serum Samples ◽

Humoral And Cellular Immunity ◽

Adenovirus Vectors ◽

Adenovirus Type 5

ABSTRACT Preclinical studies have shown that gene transfer following readministration of viral vectors is often inefficient due to the presence of neutralizing antibodies. Vectors derived from ubiquitous human adenoviruses may have limited clinical use because preexisting humoral and cellular immunity is found in 90% of the population. Furthermore, risks associated with the use of human adenovirus vectors, such as the need to immunosuppress or tolerize patients to a potentially debilitating virus, are avoidable if efficient nonhuman adenovirus vectors are feasible. Plasmids containing recombinant canine adenovirus (CAV) vectors from which the E1 region had been deleted were generated and transfected into a CAV E1-transcomplementing cell line. Vector stocks, with titers greater than or equal to those obtained with human adenovirus vectors, were free of detectable levels of replication-competent CAV and had a low particle-to-transduction unit ratio. CAV vectors were replication defective in all cell lines tested, transduced human-derived cells at an efficiency similar to that of a comparable human adenovirus type 5 vector, and are amenable to in vivo use. Importantly, 49 of 50 serum samples from healthy individuals did not contain detectable levels of neutralizing CAV antibodies.

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Tropism Modification of Adenovirus Vectors by Peptide Ligand Insertion into Various Positions of the Adenovirus Serotype 41 Short-Fiber Knob Domain

Journal of Virology ◽

10.1128/jvi.02722-06 ◽

2006 ◽

Vol 81 (6) ◽

pp. 2688-2699 ◽

Cited By ~ 23

Author(s):

Andrea Hesse ◽

Daniela Kosmides ◽

Roland E. Kontermann ◽

Dirk M. Nettelbeck

Keyword(s):

Gene Transfer ◽

Cell Types ◽

Short Fiber ◽

Peptide Ligand ◽

Cell Binding ◽

Adenovirus Serotype ◽

C Terminus ◽

Genetic Targeting

ABSTRACT Recombinant adenoviruses have emerged as promising agents in therapeutic gene transfer, genetic vaccination, and viral oncolysis. Therapeutic applications of adenoviruses, however, would benefit substantially from targeted virus cell entry, for example, into cancer or immune cells, as opposed to the broad tropism that adenoviruses naturally possess. Such tropism modification of adenoviruses requires the deletion of their natural cell binding properties and the incorporation of cell binding ligands. The short fibers of subgroup F adenoviruses have recently been suggested as a tool for genetic adenovirus detargeting based on the reduced infectivity of corresponding adenovectors with chimeric fibers in vitro and in vivo. The goal of our study was to determine functional insertion sites for peptide ligands in the adenovirus serotype 41 (Ad41) short fiber knob. With a model peptide, CDCRGDCFC, we could demonstrate that ligand incorporation into three of five analyzed loops of the knob, namely, EG, HI, and IJ, is feasible without a loss of fiber trimerization. The resulting adenovectors showed enhanced infectivity for various cell types, which was superior to that of viruses with the same peptide fused to the fiber C terminus. Strategies to further augment gene transfer efficacy by extension of the fiber shaft, insertion of tandem copies of the ligand peptide, or extension of the ligand-flanking linkers failed, indicating that precise ligand positioning is pivotal. Our study establishes that internal ligand incorporation into a short-shafted adenovirus fiber is feasible and suggests the Ad41 short fiber with ligand insertion into the top (IJ loop) or side (EG and HI loops) of the knob domain as a novel platform for genetic targeting of therapeutic adenoviruses.

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In Vitro and In Vivo Induction of Human Hematopoietic Stem Cell Migration by Extracellular UTP.

Blood ◽

10.1182/blood.v106.11.1730.1730 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1730-1730

Author(s):

Lara Rossi ◽

Rossella Manfredini ◽

Francesco Bertolini ◽

Davide Ferrari ◽

Miriam Fogli ◽

...

Keyword(s):

Cell Types ◽

P2y Receptors ◽

Extracellular Nucleotides ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Human Cd34 ◽

Cd34 Cells ◽

Stromal Cell Derived Factor

Abstract Regulatory mechanisms governing homing and engraftment of hematopoietic stem cells (HSCs) involve a complex interplay between chemokines, cytokines, growth factors and adhesion molecules in the intricate architecture of bone marrow (BM) microenvironment. HSCs express P2Y and P2X receptors for extracellular nucleotides, which activation by ATP and UTP has been recently demonstrated (Lemoli et al. Blood. 2004) to produce potent stimulatory effects on HSCs. Moreover extracellular nucleotides are emerging as key factors of flogosis phenomena and related chemotactic responses of several cell types, such as dendritic cells, monocytes and endothelial cells. In this study we investigated the biologic activity of extracellular ATP and UTP and their capacity to cooperatively promote SDF-1 (stromal cell-derived factor-1)-stimulated cell chemotaxis. Low concentrations of UTP (10uM) significantly improved, in vitro, HSCs migration. Moreover, UTP inhibits CXCR4 down-regulation of migrating CD34+ cells and increased cell adhesion to fibronectin filaments. Furthermore, in vivo competitive repopulation assays showed that preincubation with UTP significantly improved the homing efficiency of human CD34+ HSCs in nonobese diabetic/severe combined immunodeficient mice. Inhibition assays with Pertussis Toxin from B. Pertussis blocked SDF-1- and UTP-dependent chemotactic responses, suggesting that Gαi proteins may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP-treated CD34+ cells and subsequent in vitro inhibition assays with Toxin B from C. Difficile suggest that RhoGTPase Rac2 and his downstream effectors ROCK1 and ROCK2 are involved in the UTP-promoted, SDF-1-dependent HSCs migration. Taken together, our data suggest that UTP may physiologically modulate HSC migration and homing to the BM, in concert with the chemotactic peptide SDF-1, via the activation of converging signaling transduction pathways between CXCR4 and P2Y receptors, involving Gαi proteins and RhoGTPases.

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“Microenvironmental contaminations” induced by fluorescent lipophilic dyes used for noninvasive in vitro and in vivo cell tracking

Blood ◽

10.1182/blood-2009-05-224030 ◽

2010 ◽

Vol 115 (26) ◽

pp. 5347-5354 ◽

Cited By ~ 84

Author(s):

Francois Lassailly ◽

Emmanuel Griessinger ◽

Dominique Bonnet

Keyword(s):

Gene Transfer ◽

Cell Tracking ◽

Near Infrared ◽

Cross Validation ◽

Cell Types ◽

Optical Tracking ◽

Direct Cell ◽

Marked Cell

Abstract Determining how normal and leukemic stem cells behave in vivo, in a dynamic and noninvasive way, remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes, which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context, we evaluated the possibility to use gene transfer–free labeling technologies. The lipophilic dye 3,3,3′,3′ tetramethylindotricarbocyanine iodide (DiR) was selected among 4 near-infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in vivo and in vitro. We further demonstrate that all lipophilic dyes tested in this study (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine perchlorate [DiI], DiD, DiR, and PKH26) can give rise to microenvironmental contamination, including when used in suboptimal concentration, after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually, we show that this microenvironmental contamination is mediated by both direct cell-cell contacts and diffusible microparticles. We conclude that tracking of labeled cells using non–genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches.

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Subgroup B and F Fiber Chimeras Eliminate Normal Adenovirus Type 5 Vector Transduction In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.77.2.1039-1048.2003 ◽

2003 ◽

Vol 77 (2) ◽

pp. 1039-1048 ◽

Cited By ~ 43

Author(s):

John W. Schoggins ◽

Jason G. D. Gall ◽

Erik Falck-Pedersen

Keyword(s):

High Titer ◽

Adenovirus Vector ◽

Adenovirus Type ◽

Cell Types ◽

Open Reading Frames ◽

Fiber System ◽

Shaft Length ◽

Vector Targeting

ABSTRACT 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.

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