Wild-type adenovirus contains a single, 36-kb, double-stranded DNA genome flanked by inverted terminal repeats. There are over 50 serotypes, from which serotypes 2 and 5 have been most developed for use as gene-therapy vectors. This virus infects the upper respiratory tract, producing symptoms similar to those associated with colds and influenza, but as far as is known, it does not normally cause more serious disorders.
After infection, the viral DNA escapes from the lysosome and is transported to the nucleus of the cell, where it persists as an episome; multiple genomes can coexist within the nucleus of an infected cell. The adenoviral genome has eight transcriptional units, expressed in temporal sequence as early (E), intermediate (I), and late (L) genes. There are four early genes (E1–E4), encoding proteins necessary for the replication of the viral genome. E1A is the first viral gene expressed, and its product trans-activates the other promoters of early genes [20
The first-generation vectors were constructed by deleting the E1 and E3 regions of the adenoviral genome. This strategy was intended to prevent expression of the late genes, upon which viral replication depends, and provide loci into which transgenes could be cloned, usually under the transcriptional control of a heterologous promoter. Recombinant adenoviruses of this type proved to be very useful vectors, infecting a wide variety of cell types very efficiently with minimal toxicity. The utility of these vectors, however, is limited continued synthesis of viral proteins by infected cells, despite the genetic deletions. These proteins render infected cells antigenic and thus liable to elimination by the immune system, a problem exacerbated by the subsequent discovery that the E3 domain of the virus encodes immunosuppressive proteins.
Second-generation vectors have deletions in the E2 or E4 regions of the genome [21
]. These second-generation vectors are clearly improved with respect to immunogenicity and toxicity. It is unclear, however, whether these vectors' performance regarding gene expression is improved, as the inactivation of proteins encoded by E4 has been shown to impair seriously expression from heterologous promoters [22
]. In the latest versions of adenoviral vectors, all viral coding sequences have been eliminated [23
]. Production of these so-called 'gutted' vectors can be problematic and their ability to express transgenes in various tissues remains under investigation. Further improvements include the construction of adenovirus/adeno-associated virus chimeras that have the potential to provide both high transduction efficiencies and long-term transgene expression.
There are two reasons why it has proved difficult to obtain long-term gene expression with adenovirus. The first reflects the persistence of viral gene expression in cells infected with first-generation vectors. This renders the transduced cells immunogenic and thus liable to elimination by cytotoxic T lymphocytes [24
]. Moreover, it appears that adenoviruses infect antigen-presenting cells, including dendritic cells, very effectively after delivery in vivo
], contributing to the anti-adenoviral immune response. Of interest is our observation that transgene expression can persist for over a year in cells of the intervertebral discs of immunocompetent rabbits when first-generation adenovirus is used [26
]. This finding suggests that long-term gene expression is possible in cells that are non-dividing and protected from immune surveillance.
The episomal nature of genes delivered by adenoviruses is a second factor limiting the duration of gene expression. Episomal DNA is rapidly lost from dividing cells, but may be retained by nonmitotic cells. There are reports that genes delivered by gutted viruses are expressed for extended periods of time in organs such as liver and muscle, where cell division is rare.
Regardless of whether or not viral genes are expressed in transduced cells, all recombinant adenoviruses, like their wild-type parent strains, are highly antigenic. Most of us already carry antibodies to type 5 adenovirus. Furthermore, there is substantial experimental evidence that a single administration of a therapeutically useful dose of adenovirus generates a sufficient immune response to prevent successful readministration of the same vector [27
]. Strategies to overcome this include switching of serotype, transient immunosuppression, 'tolerisation' (the induction of tolerance), and attaching polyethylene glycol (PEG) moities to the virus ('PEGylation'). Gene delivery ex vivo
using a later-generation virus would also overcome problems associated with the immunogenicity of the adenovirus, but this would deprive the vector of one of its major advantages, efficient gene transfer in vivo
The antigenicity of adenoviruses not only interferes with gene delivery, but also causes pathology, usually inflammation. This has been seen after the intra-articular injection of adenovirus in mice [28
], rats [15
], and rabbits [3
], although not all authors have noted it [2
]. Some of the variation may be due to batch differences. Depending upon the preparation, only 1–10% of recombinant virions may be infectious. Although noninfectious, the other 90–99% of the viral particles are antigenic and can contribute to inflammation. The purity of the viral suspension also affects its properties, and incomplete removal of cellular debris or chemicals used in the preparation of the virus will affect performance. In addition, adenovirus may be intrinsically inflammatory as a result of its ability to activate MAP kinases and NFκB by binding to integrins on the cell surface [29
Before evaluating the future utility of adenoviral vectors in the gene therapy of arthritis, it is worth reviewing briefly the anti-arthritic strategies to which these vectors might be applied.