Replacement of Native Adenovirus Receptor-Binding Sites with a New Attachment Moiety Diminishes Hepatic Tropism and Enhances Bioavailability in Mice (original) (raw)
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Adenovirus – a blueprint for gene delivery
Current Opinion in Virology, 2021
A central quest in gene therapy and vaccination is to achieve effective and long-lasting gene expression at minimal dosage. Adenovirus vectors are widely used therapeutics and safely deliver genes into many cell types. Adenoviruses evolved to use elaborate trafficking and particle deconstruction processes, and efficient gene expression and progeny formation. Here, we discuss recent insights into how human adenoviruses deliver their double-stranded DNA genome into cell nuclei, and effect lytic cell killing, non-lytic persistent infection or vector gene expression. The mechanisms underlying adenovirus entry, uncoating, nuclear transport and gene expression provide a blueprint for the emerging field of synthetic virology, where artificial virus-like particles are evolved to deliver therapeutic payload into human cells without viral proteins and genomes.
Adenoviral vectors:: convenient tools for gene delivery to primary mammalian cells
Biotechnologia Acta, 2012
Tissue cell culture is widely used as convenient technique in modern biology and drug discovery. Multiple cell lines derived from normal and neoplastic tissues are used world-wide. However all the established cell lines, including pseudo-normal ones, are at least minimally transformed and frequently cannot properly re-capitulate the in vivo situation. Many of these limitations can be circumvented by using primary cells. Unfortunately, these cells are frequently difficult to transfect. The E1/E3-deficient recombinant adenoviruses shown to be highly efficient gene delivery tools that allow successful work with multiple types of difficult to transfect cells in different in vivo applications. Generation of the recombinant adenoviral constructs includes sub-cloning of the insert into a shuttle vector with convenient polycloning site with subsequent homologous recombination of the shuttle vector with an adenoviral backbone. We described a generation of more than 80 new rAdvs including expression, luciferase reporter and small hairpin interfering RNA vectors by using the AdEasy system. The generated recombinant adenoviruses can be used in multiple biological applications and in combination with proper maintenance protocol allow for 100% efficiency of transduction of difficult to transfect cells including primary mouse keratinocytes, human bone marrow-derived primary mesenchymal stem cells and other types of primary mammalian cells.
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.
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.
Human Adenovirus Vectors for Gene Transfer into Mammalian Cells
Advances in Pharmacology, 1997
Adenovirus (Ad) vectors are probably the most efficient means currently available for delivering foreign genes into mammalian cells both in vivo and in cell culture. The adenoviruses have been well characterized in the four decades since their first isolation, and these investigations have revealed several features of Ad biology that contribute to the effectiveness of the virus as a gene transfer vector. Numerous studies have shown that a wide variety of cell types and tissues of many different species can be infected by Ad. In addition, both dividing and nondividing cells can be infected at high efficiencies. The infection is rapid and requires no exposure to toxic or harmful substances, in contrast to some methods for introducing foreign DNA into cells. Furthermore, the genome is relatively easy to manipulate using standard molecular biological techniques, facilitating construction of recombinants. Finally, recombinant vectors can be easily produced and purified on a large scale, yielding viral stocks with titers up to lo'* plaqueforming units (PFU)/ml.
Nonreplicating Adenoviral Vectors: Improving Tropism and Delivery of Cancer Gene Therapy
Cancers, 2021
Recent preclinical and clinical studies have used viral vectors in gene therapy research, especially nonreplicating adenovirus encoding strategic therapeutic genes for cancer treatment. Adenoviruses were the first DNA viruses to go into therapeutic development, mainly due to well-known biological features: stability in vivo, ease of manufacture, and efficient gene delivery to dividing and nondividing cells. However, there are some limitations for gene therapy using adenoviral vectors, such as nonspecific transduction of normal cells and liver sequestration and neutralization by antibodies, especially when administered systemically. On the other hand, adenoviral vectors are amenable to strategies for the modification of their biological structures, including genetic manipulation of viral proteins, pseudotyping, and conjugation with polymers or biological membranes. Such modifications provide greater specificity to the target cell and better safety in systemic administration; thus, a ...
Targeted Gene Delivery to Vascular Tissue In Vivo by Tropism-Modified Adeno-Associated Virus Vectors
Circulation, 2004
Background-Gene therapy offers an unprecedented opportunity to treat diverse pathologies. Adeno-associated virus (AAV) is a promising gene delivery vector for cardiovascular disease. However, AAV transduces the liver after systemic administration, reducing its usefulness for therapies targeted at other sites. Because vascular endothelial cells (ECs) are in contact with the bloodstream and are heterogeneous between organs, they represent an ideal target for site-specific delivery of biological agents. Methods and Results-We isolated human venous EC-targeting peptides by phage display and genetically incorporated them into AAV capsids after amino acid 587. Peptide-modified AAVs transduced venous (but not arterial) ECs in vitro, whereas hepatocyte transduction was significantly lower than with native AAV. Intravenous infusion of engineered AAVs into mice produced reduced vector accumulation in liver measured 1 hour and 28 days after injection and delayed blood clearance rates compared with native AAV. Peptide-modified AAVs produced enhanced uptake of virions in the vena cava with selective transgene expression. Retargeting was dose dependent, and coinfusion of either heparin or free competing peptides indicated that uptake was principally independent of native AAV tropism and mediated via the peptide. Conclusions-AAV tropism can be genetically engineered by use of phage display-derived peptides to generate vectors that are selective for the vasculature. (Circulation. 2004;109:513-519.)
High-Capacity Adenoviral Vectors: Expanding the Scope of Gene Therapy
International Journal of Molecular Sciences, 2020
The adaptation of adenoviruses as gene delivery tools has resulted in the development of high-capacity adenoviral vectors (HC-AdVs), also known, helper-dependent or “gutless”. Compared with earlier generations (E1/E3-deleted vectors), HC-AdVs retain relevant features such as genetic stability, remarkable efficacy of in vivo transduction, and production at high titers. More importantly, the lack of viral coding sequences in the genomes of HC-AdVs extends the cloning capacity up to 37 Kb, and allows long-term episomal persistence of transgenes in non-dividing cells. These properties open a wide repertoire of therapeutic opportunities in the fields of gene supplementation and gene correction, which have been explored at the preclinical level over the past two decades. During this time, production methods have been optimized to obtain the yield, purity, and reliability required for clinical implementation. Better understanding of inflammatory responses and the implementation of methods ...
Adenovirus receptors and their implications in gene delivery
Virus Research, 2009
Adenoviruses (Ads) have gained popularity as gene delivery vectors for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral capsid proteins. Several cell surface molecules have been identified as receptors for Ad attachment and entry. Tissue tropism of Ad vectors is greatly influenced by their receptor usage. A variety of strategies have been investigated to modify Ad vector tropism by manipulating the receptor-interacting moieties. Many such strategies are aimed at targeting and/or detargeting of Ad vectors. In this review, we discuss the various cell surface molecules that are implicated as receptors for virus attachment and internalization. Special emphasis is given to Ad types that are utilized as gene delivery vectors. Various strategies to modify Ad tropism using the knowledge of Ad receptors are also discussed.