Introduction to Advenovirus Vector

1. Introduction to Advenovirus Vector The advenovirus Figure 1. Advenovirus Adenovirus is the non-enveloped double-stranded DNA, and the genome contains approximately 26-48 kbp. It was first isolated in the 1950s, but it was not until the 1980s that the therapeutic potential of adenovirus as a gene delivery vector was discovered. Adenovirus is the first viral gene delivery vector used in humans, and more than 60 types of human adenovirus have been discovered. Structural characteristics of adenovirus The complete virus particle is icosahedral with a diameter of 70-100 nm and consists of 240 hexahedral subunits (Hoxon) and 12 penton subunits (Penton base) located on the top of the regular icosahedron. Each quintuple subunit consists of a basal part and a protruding cilium (Fiber). The adenovirus genome is a linear double-stranded DNA with a 5'end covalently bound to a terminal protein (TP) and a terminal inverted repeats (ITRs) on the 5' end. Types of adenovirus vectors According to the absence of viral genes, adenovirus vectors can be roughly divided into three types: 1. First generation adenovirus vector The viral genome lacks either the E1 region or the E1&E3 region. The deletion of the E1 region results in the inability of adenovirus vectors to replicate while increasing vector capacity. Vectors lacking the E1 region can only be produced in cell lines expressing the E1 protein. However, the first generation of adenovirus vectors may produce replicative viruses due to homologous recombination. 2. Second generation adenovirus vector The disadvantages of the first-generation adenovirus vectors are minimized. The second-generation viral vectors simultaneously delete E1&E3&E2/E4. This does reduce the immunogenicity of the vector and also increases the vector capacity, which is larger than the first generation. But this leads to the need for an appropriate cell to ensure the replication and reproduction of the virus. 3. Third generation adenovirus vector A helper plasmid relies on a viral vector that removes all adenovirus genes, retaining only the ITR and packaging signals of the viral genes. It significantly reduces the immunogenicity, making it more suitable as a gene delivery vector. Its carrier capacity

2. becomes larger than the first and second generation. At the same time, the cre/loxp system is used for the auxiliary plasmid, so that the generated auxiliary plasmid viral genome does not contain packaging signals and is more safe. Third-generation adenovirus vectors have minimal side effects in different animal models. Advantages of adenoviral vectors 1. Wide host range and low pathogenicity to human Adenovirus vector systems can be widely used for the expression of human and non- human proteins. Adenovirus can infect a range of mammalian cells, so they can be used to express recombinant proteins in most mammalian cells and tissues. In particular, it should be noted that adenovirus is epitheliophilic, and most human tumors are of epithelial origin. In addition, the replication genes and pathogenic genes of adenovirus are quite clear and have been prevalent in the population for a long time. 2. Infection and expression of genes in proliferating and non-proliferating cells Retroviruses can only infect proliferative cells, so DNA transfection cannot be carried out in non-proliferative cells, and cells must be kept in a state of continuous culture. Adenovirus can infect almost all cell types, except some lymphoma cells that are resistant to adenovirus infection. Adenovirus is the best system for studying gene expression in primary non-proliferating cells, which allows direct comparisons between the results obtained in transformed and primary cells. 3. They can effectively proliferate with a high titer The adenovirus system can produce 1010 to 1011 VP/ml, which can be concentrated to 1013 VP/ml, which makes it very suitable for gene therapy. 4. Unconformity into chromosomes, no insertion mutagenicity Retrovirus can be randomly integrated into host chromosomes, leading to gene inactivation or activation of oncogenes. Adenoviruses, on the other hand, do not integrate into chromosomes in almost all known cells except the egg cells, and therefore do not interfere with other host genes. Integrating single-copy viruses in egg cells is a better system for generating genetically modified animals with specific characteristics. 5. They can express multiple genes at the same time This is the first expression system that can be designed to express multiple genes in a uniform cell line or tissue. Disadvantages of adenovirus vectors 1. Inability to express for a long time; 2. High immunogenicity, especially at high doses, may cause severe toxicity. Adenovirus vector packaging methods There are currently two approaches: 1. Single plasmid form, i.e. inserting foreign genes directly into viral genomic plasmids using specific endonucleases; 2. The double plasmid form is to use homologous recombination to complete recombination in bacteria or cell lines. Two plasmids are encapsulated in double plasmid forms: genomic plasmid and shuttle plasmid. Genomic plasmids are appropriately deleted and contain almost the entire adenovirus genome. Shuttle

3. plasmids contain foreign genes as well as key genomic sequences that ensure successful homologous recombination and the generation of viruses with infectious capacity.