Adenoviral vectors are produced in the 293 cell line, which provides in trans the E1 functions that render them condition ally replication competent, permitting vector growth. The difficulty with this approach to propagate vectors is that there is the possibility of homologous recombination between the replication-deficient vector and the chromosomal copy of the Ad5 genome (Fig. 6) (32). This inevitably occurs at a low frequency, resulting in the production of E1 + , E3~ replication-competent adenovirus (RCA). Once formed, RCA will outgrow the replication-deficient gene therapy vector in vitro. To minimize the production of RCA, Ad are plaque purified several times on 293 cells and exhaustively tested for the presence of RCA, which is readily detected on the basis of its ability to form plaques on E1 negative cell lines such as the human lung epithelial cell line A549 (32). For clinical studies, adenoviral vectors should be uncontaminated by RCA (level <1 RCA per dose). However, the preparation of Ad vector of this quality is difficult and most in vivo animal and in vitro
Figure 6 Production of replication-competent adenovirus by homologous recombination between Ad vector and genome of 293 cells. The 293 cell line (open rectangle) contains nucleotides 1 through 4344 from adenovirus type 5, including the left inverted terminal repeat (LITR), the E1A and E1B genes, and the adjacent protein IX gene (pIX). The E1 deletion in most first-generation vectors (top rectangle) stretches from nucleotide 355 through 3328, which is replaced by the expression cassette for the therapeutic gene. Therefore, 2 homologous recombination events (crossed line) can occur that restore the E1 region and give a replication competent (albeit E3 ") virus. In the second example, the extent of the E1 deletion in the vector has been extended to encompass all the E1A and E1B genes. At the same time, the E1A and E1B genes in the complementing cell line [i.e., perC6 cells (159)] have no flanking sequence and expression is driven by the phosphoglycerol kinase promoter (p PGK). As a result, there is no homology at either end and only 2 illegitimate recombination events can result in the production of RCA. Therefore, the frequency is very low.
studies are done with preparations with uncharacterized levels of RCA that are probably >1 RCA per 108 particles.
To reduce RCA production, two approaches can be used: reduce the size of the trans-complementing E1 region in the cell line or increase the size of the E1 deletion in the vector. Several cell lines have been developed that have less of the Ad genome (compared with 293 cells), while retaining the ability to supply the E1A and E1B functions in trans and the high productivity of the 293 cell line (Table 1). The E1 deletion in the first-generation clinical vectors was smaller than optimal, retaining 31% of the 3' end of the E1B gene. Deleting this sequence in conjunction with cell lines that express E1A/ B from nonadenoviral promoter allows production of adeno-viruses in circumstances where there is no overlap between the cellular sequence and the vector (Fig. 6B). In these cell lines, RCA is virtually eliminated because it can only arise through two illegitimate recombination events, an occurrence that is very rare.
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