**4. Detargeting Ads**

**4. Detargeting Ads** Adenovirus has not evolved as a cancer selective pathogen, and therefore requires engineering in order to effectively target cancer cells. In Ad-based therapies, the efficiency of the treatment can be greatly affected by their native binding interactions. The most studied Ad, Ad5, binds hCAR which localises to the tight junctions between cells and is expressed ubiquitously throughout the body [33,118]. hCAR has also been reported as downregulated in certain cancers [119–121]. Therefore, reliance upon hCAR as an entry receptor for any Ad-based cancer therapy would greatly limit its uses, as transduction would be limited to cancer cells with high-hCAR expression, and off-target transduction could lead to tissue toxicity. Moreover, more aggressive cancer growth correlates with loss of hCAR expression, and so non-targeted Ad therapy is unlikely to treat aggressive cancers Adenovirus has not evolved as a cancer selective pathogen, and therefore requires engineering in order to effectively target cancer cells. In Ad-based therapies, the efficiency of the treatment can be greatly affected by their native binding interactions. The most studied Ad, Ad5, binds hCAR which localises to the tight junctions between cells and is expressed ubiquitously throughout the body [33,118]. hCAR has also been reported as downregulated in certain cancers [119–121]. Therefore, reliance upon hCAR as an entry receptor for any Ad-based cancer therapy would greatly limit its uses, as transduction would be limited to cancer cells with high-hCAR expression, and off-target transduction could lead to tissue toxicity. Moreover, more aggressive cancer growth correlates with loss of hCAR expression, and so non-targeted Ad therapy is unlikely to treat aggressive cancers through hCAR transduction alone [118].

through hCAR transduction alone [118]. hCAR binding can be ablated by mutating the key amino acids in the fibre knob AB loop (L5 gene), using the KO1 mutations, S408E and P409A [66,122]. hCAR mediated cell entry is a two-step mechanism. First the virus attaches to hCAR and, secondly the virion internalised through binding of an Arg-Gly-Asp (RGD) motif in the penton base to αvβ3/αvβ5 transmembrane integrins on the cell surface [123]. This secondary interaction has been exploited to further detarget Ad5 through the RGD to RGE modification of the penton base and improve cancer targeting. The Ad5NULL vector hCAR binding can be ablated by mutating the key amino acids in the fibre knob AB loop (L5 gene), using the KO1 mutations, S408E and P409A [66,122]. hCAR mediated cell entry is a two-step mechanism. First the virus attaches to hCAR and, secondly the virion internalised through binding of an Arg-Gly-Asp (RGD) motif in the penton base to αvβ3/αvβ5 transmembrane integrins on the cell surface [123]. This secondary interaction has been exploited to further detarget Ad5 through the RGD to RGE modification of the penton base and improve cancer targeting. The Ad5NULL vector encompasses the KO1 mutation, the RGD to RGE modification as well as a modification within

encompasses the KO1 mutation, the RGD to RGE modification as well as a modification within the

the hexon hypervariable region (HVR7) in order to ablate binding to coagulation FX and prevent sequestration by and transduction of liver hepatocytes [13,124].

Larger modifications can also be made to detarget the virus particle, such as replacing the fibre shaft of Ad5 with shorter variations found in other serotypes such as Ad40, or Ad41. This approach has shown reduced binding to cells [125–127].

Chemical modifications can also be used as a means of detargeting through polymer coating of the Ad particle. Polyethylene glycol (PEG) is commonly used for this purpose, due to its cationic properties [128]. The main advantage of coating the Ad is to prevent neutralisation by pre-existing antibodies which reduces the efficiency in patients previously exposed to the Ad. The use of chemical modifications was well reviewed elsewhere previously by Kreppel and Kochanek [129] and Kim et al. [130]. A major disadvantage of non-genetic means of targeting is that daughter virions produced through replication will not harbour the modifications necessary to target tumour cells, and therefore genetic strategies which are heritable are therefore more commonly preferred.
