Brief Introduction of Recombinant Adenoviruses

Adenoviruses (ADV) are non-enveloped particles containing a linear double-stranded DNA of ~36 kb. Recombinant adenovirus (rADV) is engineered to contain gene of interest and have been considered as an attractive tool for gene delivery.

ADV has several features that make it possible to be designed as a useful genetic transfer tool. 1) Like LV, ADV can transduce both dividing and non-dividing cells with almost 100% transduction efficiency. 2) Compared with lentivirus (LV) and Adeno-associated Virus (AAV), ADV can accommodate larger transgenes. 3) Optimized protocols have been developed for producing rADV at high titers. 4) The viral genome of ADV does not integrate into the host cells’ genome which means ADV can not cause random insertion mutation but also means it can not be used for long-term expression but for transient expression.

There is a known drawback using ADV which is its strong immunogenicity that may lead to the death of transduced cells. Interestingly, scientists have also utilized this drawback to develop rADV vectors for applications in oncolysis and vaccination.

rADVs can be divided into different subgroups according to the fiber protein, cloning capacity and replication ability.

1) Subgroups based on fiber protein
More than 50 serotypes of human ADVs have been identified. Human adenovirus serotype 5 (Ad5) has been well characterized and the most commonly used rADVs are based on human Ad5. Ad5-based rADVs infect target cells through coxsackie-adenovirus receptor (CAR). However, some cells have no expression or low expression of CAR resulting inefficient Ad5-based transduction. To overcome this limitation, several types of fiber-modified rADVs have been developed and effectively used, such as Ad5/35, Ad5/RGD. Ad5/35 is a chimeric rADV consisting of the knob and shaft of Ad35 combined with Ad5. Ad5/35 attaches to cells through a different cell receptor CD46 allowing efficient transduction of CAR-negative cell lines. Ad5/RGD is another type of chimeric rADV consisting of Arg-Gly-Asp (RGD) motif in the surface-exposed loops of Ad5 fiber knob. Ad5/RGD mediates cell entry via RGD binding integrins.

2) Subgroups based on cloning capacity
Three generations of rADVs have been developed which are first-generation rADVs (FG-rADVs), second-generation rADVs (SG-rADVs) and Third-generation rADVs (TG-rADVs). FG-rADVs are engineered by deleting E1/E3 region, often lacking E1A and E1B, for transgene insertion. SG-rADVs contains more deletions including E1/E3 and E2/E4 early regions which decrease the risk of replication-competent adenovirus (RCA) formation and increase the capacity for exogenous DNA. TG-rADVs, also known as helper-dependent rADVs (HD-rADVs) or gutless rADVs (GL-rADVs), lacks almost all adenoviral sequences except for sequences that are essential for packaging, such as cis-acting inverted terminal repeat (ITR) sequences which are required for viral DNA replication and a cis-acting packaging signal (Ψ) which is required for the encapsidation of the viral genome. Unlike FG-rADVs and SG-rADVs, TG-rADVs lack adenoviral genes that are essential for the production of virus particles, therefore, production of TG-rADVs requires helper adenoviruses providing the missing elements. Compared with FG-rADVs and SG-rADVs, TG-rADVs possess even greater cloning capacity allowing insertion of multiple transgene expression cassettes.

Figure 1. Schematic representation of wild-type adenovirus genome and the different generations of nonreplicating AdV. E1 to E4, early-region transcript units; L1 to L5, late region transcript unit; ITR, inverted terminal repeats; MLP, major late promoter; Ψ, packaging signal, GOI, gene of interest. [Adenoviral Vectors for Gene Therapy, David T. Curiel · 2016]

3) Subgroups based on replication ability
Based on the replication ability, there are two types of rADVs, replication-deficient rADVs and replication-competent rADVs. The E1 gene products, including E1A and E1B, play roles in the replication of adenoviruses. Most rADVs are both E1 and E3 deleted (dE1/dE3) and can not replicate in cells lacking E1 gene products but can replicate in cells containing complementary E1 gene products (e.g., HEK293). Some rADVs remain intact E1A region and therefore can replicate in any transduced cells. Oncolytic adenoviruses (OAds) belong to replication-competent rADVs and have been explored extensively as a viral vector for gene therapy.

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