Vectors Based on Adenoviruses
Adenoviral vectors are the most commonly used vector in clinical trials and attractive candidates for the transfer of foreign genes. They have shown considerable application promise as delivery vehicles in gene therapy and vaccination.
Biological Properties of Adenoviruses
Adenoviruses are non-enveloped, linear double-stranded DNA viruses, in size from ~70 to 100 nm. The virion consists of an icosahedral capsid, made up of subunits hexon proteins, penton proteins, and knobbed fiber proteins, surrounding an inner viral DNA core, which contains the genomic material of the virus.
Generally, the adenoviral genome is a linear, dsDNA molecule of about 34-43 kb, a size that is amenable for easy manipulation, and the entire genome encodes about 30 proteins. Both extremities of the viral genome are flanked by the inverted terminal repeats (ITR) of varying lengths, which serve as the origin for DNA replication. The adenoviral genome is grouped into five early (E1-E5) transcriptional units encoding regulatory proteins for viral replication, and five late (L1-L5) genes encode structural proteins, which are essential for the assembly.
Figure 1. The general adenovirus structure. (Zhang, 2006)
Adenoviruses are species-specific, and approximately 50 known serotypes have been isolated from human adenoids. Based on genome size, composition and homology, adenoviruses are divided into six subgroups, from A to F. Among these groups, adenovirus type 5 (Ad5) from group C has been the most studied to use for gene therapy.
Classification of Adenovirus Vectors
To date, adenoviral vectors have gone through several steps of development from the first-generation to third-generation vectors and exploited mainly for gene therapy. Although early versions of adenoviruses showed toxic side effects and robust immune responses, newer second-generation and third-generation vectors with many of the viral genes deleted are demonstrated significant improvements to prolong transgene expression with reduced immune reactions.
Figure 2. Organization of the adenovirus genome. (Tatsis, 2004)
- E1-deleted Adenovirus Vectors (First-Generation)
Most vectors currently used in preclinical development and clinical trials for gene transfer applications are based on the first-generation replication-incompetent vectors (FGV), they are generated replication-defective by deletions of E1 that are essential for viral replication and expression. In order to further increase the transgene insertion capacity, many vectors contain partial deletions within the E3 region of the genome, which is dispensable for replication and packaging, then replacing them with transgene expression cassettes. They provide sufficient space for gene insertions and allow the introduction to up 8.3 kb of foreign DNA into the E1, E3 regions. However, this generation of vectors retains many of the viral genes resulting in the low levels of DNA replication and the limitation of the duration of therapeutic gene expression. This leads to the development of second and third generation adenoviral vectors.
- Vectors with Additional Mutations in Other Viral Genes (Second-Generation)
More extensive defective adenoviruses as second-generation vectors are established by bearing additional deletions in the E2 and/or E4 regions of adenovirus. These vectors allow extra space for the insertion and could accommodate up to 14 kb of foreign DNA. However, these vectors did not completely solve the issue of adenovirus induced toxicity, and the duration of gene expression also appears to be reduced. So far, second-generation vectors have not been widely distributed, mainly because of difficulties during production.
Figure 3. Different types of adenovirus vectors. (Robbins, 1998)
- Helper-Dependent Gutless Adenovirus Vectors (Third-Generation)
In an attempt to overcome the first and second-generation potential issues, the third-generation adenoviral vectors are constructed by the complete deletion of the most adenoviral coding regions replacing by the transgene(s) of interest except for the ITRs and the packaging sequence; thus, the vectors are termed "gutless" or more appropriately, helper-dependent adenoviruses vectors (HD-Ad). The significant advantages of this system are the viral replication under control, and enough space to accommodate up to 36 kb of transgene sequence into the vector genome. It allows to delivery of large DNA sequences or multiple genes, and significantly improves performance and reduces toxicity and immunogenicity at the same time.
Advantages of Adenovirus Vectors
- Easy to produce in high functional viral titers (1011-1013 PFU/ml)
- A large packaging size and transgene capacity up to 36 kb
- Efficient transduction of target cells independent of cell-cycle
- A broad tropism and can infect both dividing and nondividing cells
- Ensure high levels of transient transgene expression
- Well-characterized and comparatively easy to manipulate
- Well suited as oncolytic vectors
- Increased predictability and reduced unwanted side effects
Application of Adenovirus Vectors
Overall, there is no question that adenovirus vectors are the most effective means of delivering genes to most organs in vivo and used as vaccine vectors to treat or prevent infections. While the immunogenicity of the adenovirus as a gene transfer vector and low-level expression of adenovirus genes limit the length of expression time to about two weeks, adenovirus vectors can be better suited for leveraged as ideal vectors in vaccination or in treating diseases for which only the short time of expression and purposefully evoking immunity is required, such as cardiovascular diseases, and cancer. Over the years, adenovirus-based vectors, particularly those derived from HAd5, have been tested in several clinical trials and are attractive as useful platforms despite their current drawbacks, mainly not only as cancer therapeutics but also as therapeutic and prophylactic agents against other indications, including infectious diseases.
References
- Zhang, X.; Godbey, W.T. (2006). Viral vectors for gene delivery in tissue engineering. Advanced drug delivery reviews. 58(4): 515-534.
- Tatsis, N.; Ertl, H.C.J. (2004). Adenoviruses as vaccine vectors. Molecular Therapy. 10(4): 616-629.
- Robbins, P.D.; Ghivizzani, S.C. (1998). Viral vectors for gene therapy. Pharmacology & therapeutics. 80(1): 35-47.
