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The adenovirus, an 80-100 nm, icosahedral capsid, double-stranded DNA virus, is a well-known cause of respiratory tract infections (Figure 1). Adenovirus infection in humans generally is mild, but in immunocompromised individuals, it can be life threatening. In the late 1980s, when researchers began to think about strategies of transferring genes in vivo, the adenovirus was known to be trophic for the respiratory epithelium. The virus had been sequenced in its entirety, a number of human serotypes were known, and the detailed biology of how the virus replicated and assembled was well described. The stage was set, partly by accident, to adapt the adenovirus to be an effective method of transferring genes in vivo.
Figure 1. Adenovirus capsid consists of three major and four minor proteins.
Adenovirus and Gene therapy
Gene therapy and virotherapy are approaches to introduce therapeutic genes into cancer cells for the treatment of cancer. The adenoviral vector has been used as a transfer vehicle to introduce genes into cancer cells because it is more efficient than non-viral gene transfer methods (for example, cationic polymer-DNA complexes). The adenoviral vector is efficient in gene delivery to both dividing and non-dividing cells and rarely causes any significant disease itself. Nevertheless, one of the limitations of gene therapy using the adenoviral vector is the non-specific expression of therapeutic genes in normal cells causing toxicity to non-cancerous tissues. Targeted expression of therapeutic genes is crucial to prevent this toxicity. Recent understanding of tissue- and cancer-specific gene regulation in organogenesis and cancer fields has enabled the development of new enhancer or promoter systems to express therapeutic genes only in targeted cells and tissues. Cancer-specific therapeutic gene expression reduces undesirable toxicity. Therefore, adenoviral vectors carrying a cancer-specific promoter system driving a therapeutic gene that is expressed only in cancer cells could be a means to safeguard against unwanted transduction to normal tissues and cells.
The early adenoviral vectors targeted not only cancer cells, but also normal cells since constitutive promoters were used to drive therapeutic genes. The expression of therapeutic genes in unintended, non-targeted normal tissues likely causes undesirable toxic side effects. In order to reduce this toxicity, cancer or tissue-specific promoter systems have been developed by replacing constitutive promoters with promoters or enhancers of tissue/cancer-specific gene markers in the adenovirus vector. These tissue-specific promoter systems can target cancers originating from different tissues including breast, prostate, lung, myeloma, and pancreatic cancers.
In early gene therapy attempts for the treatment of cancer, viral vectors were used that were modified to delete the function of self-replication to achieve safe gene transfer without inducing viral lysis to normal tissues and cells. Nevertheless, the clinical trials of some suicide gene or corrective gene therapies revealed that the therapeutic effect of non-replicative viruses was limited. In order to resolve this limitation, conditional replicative adenoviruses (CRAds) were developed for cancer gene therapy, which is also called virotherapy. The conditional replicative viruses can replicate and cause cell lysis only in the targeted tumor cells. Furthermore, the replicated viruses can also infect neighboring cancer cells and continue this infection cycle until all of the tumor cells are eradicated.
Oncolytic adenoviruses (Ads) have attracted a great deal of interest as cancer therapeutics since they can be genetically manipulated and exhibit various distinct anticancer mechanisms. Not only do oncolytic Ads directly kill tumor cells at the end of their lytic cycle (Figure 2), but also progeny viruses spread throughout a tumor, infecting other cancer cells. Based on these characteristics, oncolytic Ads are considered effective in treating bulky tumors and potentially metastatic disease. Even as these first-generation oncolytic Ads have been tested clinically, innovative genetic engineering approaches have proposed designs for several second- and third-generation oncolytic Ads that demonstrate enhanced therapeutic efficacy in neoplastic tissue. In addition, so-called “armed” oncolytic Ads have been developed to deliver therapeutic genes, further expanding the potential antitumor mechanisms carried by a single vector.
Figure 2. Schematic diagram of the cancer-selective killing efficacy of oncolytic Ads
Major challenges of cancer-targeting vectors
One of the major challenges in adenovirus-mediated cancer gene therapy is poor transduction in human tumors. The reason for this poor transduction is that tumor cells have limited surface expression of the Ad5 primary receptor, the coxsackievirus and adenovirus receptor (CAR) which are necessary for transduction. In order to resolve the problem, several modifications to the adenovirus fiber have been performed. Wickham et al. constructed adenoviral vectors, which contain modifications to the adenoviral fiber coat protein that redirect virus binding to either α(v) integrin [AdZ.F (RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. The AdZ.F (RGD) can increase gene delivery to endothelial and smooth muscle cells expressing α(v) integrins and AdZ.F (pK7) increases transduction 5- to 500-fold in multiple cell types lacking high levels of the adenoviral fiber receptor. Another approach is to create chimeric viruses, usually based on adenovirus serotype 5 (Ad5). The fiber/knob domain is replaced by that of another serotype Ad5 with adenovirus serotype 3 (Ad3). This virus showed CAR-independent infectivity. Ulasov et al. have shown that a chimeric Ad5/3 vector which contains the shaft of adenovirus serotype 5 and the knob of adenovirus serotype 3 can target the CD46 cellular receptor and increase transduction of glioma cells. Kanerva et al. reported that the Ad5/3 vector increased transduction of ovarian cancer cells. Similarly, adenoviral vectors constructed with the serotype 17 fiber (Ad17) can improve transduction of airway epithelial tissue.
Adenoviruses have been crucial tools to explore issues of tropism and regulatable expression and, importantly, opened the door to effective clinical gene therapy. For short-term, high-level expression or for purposefully evoking immunity, the adenovirus vectors stand alone as the choice for the gene therapist. Recently, researchers have been working to improve the efficacy of these vectors by focusing on improving three parts of the vector systems. One goal is to obtain specificity to cancer cells to avoid damaging normal cells. A second goal is to improve the means to induce cancer cell death. A third goal is to improve transduction. The surface structure of adenovirus vectors has been modified to deliver therapeutic genes into cancer cells more efficiently. In addition, some of the modifications enable the virus to avoid the immune response of the patient.