”1.

The delta24 (∆24) deletion results in specific replication in tumor cells. During wild type adenovirus replication in normal cells, the adenoviral E1A gene product forms a complex with the host cell retinoblastoma tumor suppressor protein (pRb), thereby releasing the cell cycle transcription factor E2F-1. This forces the cell into cell cycle (S-phase) and promotes replication of the adenovirus by driving expression of the viral E2 gene region. Conversely, loss of this E1A function renders the adenovirus replication incompetent in normal cells with a functional pRb pathway. A simple deletion of 24 base pairs in the CR2 region of E1A abolishes the binding of E1A to pRb. Therefore, viruses with this type of deletion are reduced in their ability to overcome the G1-S checkpoint and replicate efficiently only in cells where sequestration of pRb by E1A is not necessary, e.g. in tumor cells, the majority of which are defective in the pRb/p16 pathway (Fueyo et al. Oncogene 2000; Heise et al. Nature Medicine 2000). Hence, viruses containing the ∆24 deletion preferentially replicate in and kill tumor cells.

”2.

Normal adenovirus entry is mediated by initial binding of the adenovirus fiber protein to its primary receptor, the Coxsackie-adenovirus receptor (CAR). The RGD-4C motif is a 9-amino acid peptide (CDCRGDCFC) that binds to integrins (particularly αvβ3 or αvβ5). Insertion of this RGD motif in the HI loop of the fiber protein of adenovirus results in enhanced infectivity in a number of different cancer cell lines (Dimitriev et al. Journal of Virology 1998; Suzuki et al. Clinical Cancer Research 2001).

”3.

The T1 mutation refers to a specific mutation resulting in enhanced viral release from infected human cancer cells and cancer-associated fibroblasts in vitro, and in enhanced antitumor activity when injected intratumorally or systemically in different models in vivo (Gros et al. Cancer Research 2008). The T1 mutation is a single Adenine insertion at position 445 of the nucleotide sequence of the E3/19K gene of Adenovirus, which was identified after natural selection of random mutated adenoviruses in tumor xenografts. This insertion causes a frame shift in the open reading frame of the E3/19K protein, and creates a truncated protein that is relocated to the plasma membrane due to the disruption of its ER retention signal. The relocation of the E3/19K protein was demonstrated to increase plasma membrane permeability and to cause faster release of the virus. Hence, incorporation of the T1 mutation in oncolytic adenovirus results in improved spread through the tumor.

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