Multiply Deleted Adenoviral Vectors

Although the deletion of E1 in FGAds theoretically results in a replication-defective vector following transduction of cells devoid of trans-complementing E1 functions, it has become clear that this is not the case and that the E2, E3, and E4 promoters are active resulting in viral DNA replication and expression of the late viral genes, especially following high multiplicities of infection (27-30). Therefore, in an attempt to further attenuate Ad, additional deletions of essential viral genes have been pursued. Examples of these include deletion or mutation of the E2 or E4 regions in addition to E1. These multiply deleted Ad vectors (also referred to as second- or third-generation Ad vectors) are helper virus independent for propagation but require the generation of new producer cell lines that trans-complement the additional deletion. Despite the potential offered by these multiply deleted Ad vectors, viral coding sequences still remain and therefore so does the potential for their expression. The advantages of multiply de

Figure 1 Transcription map of human adenovirus serotype 5. The 100 map unit (—36 kb) genome is divided into 4 early region transcription units, E1-E4, and 5 families of late mRNA, L1-L5, which are alternative splice products of a common late transcript expressed from the major late promoter located at 16 map units. Four smaller transcripts, pIX, IVa, and VA RNAs I and II, are also produced. The 103 bp inverted terminal repeats (ITRs) are located at the termini of the genome and are involved in viral DNA replication, and the packaging signal located from nucleotides 190 to 380 at the left end is involved in packaging of the genome into virion capsids.

leted Ad over FGAd remain controversial because some studies show them to be superior in terms of toxicity and longevity of transgene expression (31-37), whereas others do not (21,38-41). Detailed discussion of multiply deleted Ad vectors is beyond the scope of this chapter; therefore, the reader is referred to an excellent and comprehensive review covering this subject by Parks and Amalfitano (42).

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