Adenovirus (Ad) vectors have many desirable characteristics which have allowed them to become popular gene transfer vehicles (2
). Many of the gene therapy “successes” when using Ads in animal models involve transduction of the liver either to restore a functional deficiency to hepatocytes or to use this organ as a protein production factory to produce large amounts of secreted protein. There is a practical reason for this: when Ad is injected systemically, greater than 80% of the virus is retained in the liver (27
). While this may be an advantage in many studies, it is one of the limitations to effective Ad therapy in many other disease models for which tissue-specific gene expression is required. Nonspecific vector transduction is undesirable for several reasons. First, less vector is available to interact with the target tissue, necessitating higher doses of Ad to achieve a given level of therapeutic protein expression. Second, acute toxicity caused by Ad is, at least in part, due to activation of the innate immune response, possibly mediated by Kupffer cells of the liver (62
). Third, promiscuous vector transduction can include infection of antigen-presenting cells which will enhance the formation of antivector and antitransgene immune responses (31
). Thus, the development of novel strategies which lead to greater efficiency and specificity of infection of target tissue and reduced infection of nontarget tissues is required.
The majority of gene therapy studies utilize Ad vectors based on serotype 2 or 5 (Ad2 or Ad5, respectively). Ad5 infection initiates with the capsid protein fiber binding to the cell surface coxsackie-adenovirus receptor (CAR) (4
), followed by a secondary interaction between penton protein and av
integrins, which triggers internalization of the virus by endocytosis (81
). Other studies have suggested that Ad can enter cells by using heparan sulfate proteoglycans as an alternative receptor through a bridging interaction between Ad and blood factors such as factor IX and complement component C4-binding protein (66
). Recently, the mechanism of high-efficiency uptake of Ad by the liver has been elucidated: the virus hexon capsid protein binds to blood factor X, which then interacts with heparan sulfate on the surface of hepatocytes (33
). Importantly, swapping the hypervariable regions of the Ad5 hexon with those of Ad48, which does not interact with factor X, reduces hepatocyte uptake 600-fold. This later work represents a paradigm shift in our understanding of Ad infection in vivo
and clearly shows that detargeting Ad can circumvent the problem of liver sequestration.
There are two main strategies for retargeting Ad infection specificity: covalent or noncovalent attachment of targeting ligands to the capsid and genetic modification of capsid proteins. Covalent and noncovalent methods involve the addition of targeting ligands after the virus has been purified, through the use of bispecific antibodies (one binding the Ad virion and the other binding the desired cellular ligand) or antibody-receptor ligand complexes or by mixing of chemically modified Ad virions with a reactive ligand (37
). Alternatively, genetic modification involves cloning of the targeting ligand directly into one of the virion coat proteins (19
). These two strategies have been combined to produce a metabolically biotinylated vector that can be combined with a variety of targeting ligands to achieve cell-type-specific targeting (9
). Since the natural protein for virus attachment to the cell is the capsid fiber protein, many groups have focused upon genetic modification of this protein in order to redirect virus attachment (24
). Targeting moieties placed on fiber can be combined with other mutations that abolish binding to the native cellular receptor (30
), thereby reducing Ad promiscuity as well as redirecting binding.
Other capsid proteins besides fiber can also be modified and used for virus retargeting. Incorporation of an arginine-glycine-aspartic acid (RGD) motif into the hexon of Ad resulted in enhanced transduction of cells expressing low levels of CAR (79
). The Ad penton protein was modified to incorporate a FLAG epitope tag, and this virus was subsequently used in a bispecific-antibody strategy (82
). Minor capsid protein IX (pIX) has also served as a platform for presentation of targeting ligands, including a polylysine motif (20
), an RGD motif (78
), or a biotin acceptor peptide for subsequent addition of targeting ligands (10
). Taken together, these studies demonstrate that the Ad capsid can be modified through several different proteins to redirect Ad binding to the cell type of interest.
We and others have shown that pIX can be modified genetically to incorporate large polypeptides into the Ad capsid (38
). pIX is a minor capsid protein that stabilizes the hexon on the facets of the capsid (14
). Recent studies showed that the N-terminal domains of three pIX monomers form a triskelion structure that cements three hexon proteins together, whereas the C-terminal domains are located near the edge between two facets and form a tetramer (22
). Three of the four C-terminal domains associate together in a parallel structure, whereas the fourth domain, which stretches across from one facet to the adjacent facet, associates with the trimer in an antiparallel manner. Capsids which contained a pIX-green fluorescent protein (GFP) fusion showed normal growth characteristics and could be visualized by fluorescence microscopy both in vitro
and in vivo
). Curiel and coworkers (39
) used pIX for the attachment of luciferase and herpes simplex virus (HSV) thymidine kinase for visualization of virus in vivo
and to generate a single virus containing three different pIX isoforms displaying a FLAG tag, 6×His tag, and monomeric red fluorescent protein (RFP) (74
). Hoeben and colleagues (18
) showed that pIX fused to a hyperstable antibody directed to β-galactosidase or a single-chain T-cell receptor targeted to MAGE-A1 antigen was efficiently incorporated into the Ad capsid and bound its ligand. Other research groups have used pIX to display complement-inhibiting polypeptides (64
). Finally, C-terminal cysteines added to pIX have been used as a target for the chemical addition of targeting ligands bearing reactive thiol groups (15
The ability to fuse large polypeptides to pIX suggests that it may serve as a platform for the addition of other large targeting ligands, such as single-chain variable-fragment antibodies (scFv) or single-domain antibodies (sdAb) (17
), which are likely capable of providing greater specificity of infection than many previously utilized, smaller ligands. scFv are heterodimers of the variable light (VL
) and heavy (VH
) chains of an antibody joined by a peptide linker and retain the binding specificity of a monoclonal antibody. sdAb are derived from camels and llamas and are formed by heavy chains only; the antigen-binding site of these antibodies consists of one single domain (VHH
). scFv and sdAb have exquisite specificity and high antigen-binding affinity, and many different antibodies that recognize surface receptors on different cell types have been described (3
). Presentation of scFv or sdAb on the capsid of Ad should improve the specificity with which Ad infects a target cell. In this study, we describe our efforts designed to redirect Ad infection specificity through display of scFv and sdAb on the surface of the Ad capsid by genetic fusion to the pIX capsid protein.