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.2010 Aug;2(8):1681-1703.
doi: 10.3390/v2081681. Epub 2010 Aug 16.

Adenoviral producer cells

Affiliations

Adenoviral producer cells

Imre Kovesdi et al. Viruses.2010 Aug.

Abstract

Adenovirus (Ad) vectors, in particular those of the serotype 5, are highly attractive for a wide range of gene therapy, vaccine and virotherapy applications (as discussed in further detail in this issue). Wild type Ad5 virus can replicate in numerous tissue types but to use Ad vectors for therapeutic purposes the viral genome requires modification. In particular, if the viral genome is modified in such a way that the viral life cycle is interfered with, a specific producer cell line is required to provide trans-complementation to overcome the modification and allow viral production. This can occur in two ways; use of a producer cell line that contains specific adenoviral sequences incorporated into the cell genome to trans-complement, or use of a producer cell line that naturally complements for the modified Ad vector genome. This review concentrates on producer cell lines that complement non-replicating adenoviral vectors, starting with the historical HEK293 cell line developed in 1977 for first generation Ad vectors. In addition the problem of replication-competent adenovirus (RCA) contamination in viral preparations from HEK293 cells is addressed leading to the development of alternate cell lines. Furthermore novel cell lines for more complex Ad vectors and alternate serotype Ad vectors are discussed.

Keywords: Ad vectors; RCA; adenovirus; producer cell lines.

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Figures

Figure 1.
Figure 1.
Genome structures of first and second generation and high capacity adenovirus vectors. Promoters are indicated by black triangles, early and late transcription units are indicated by thick and thin arrows respectively. The inverted terminal repeats (ITR) are indicated by black squares and the packaging signal, Ψ is indicated. Non-adenoviral sequences in the high capacity genome are indicated by a thin line, while transgenes in all genomes are indicated by diagonal striped boxes.aA very large number of different systems are in use with specific extra capacity. For second generation vectors deletion of 3 gene loci and a conservative 103% genome size was used for these estimates.
Figure 2.
Figure 2.
RCA generation in HEK293 cells. The complementary regions of Ad5 sequence in the vector and the HEK293 genome are aligned at the top of the figure. These regions allow for homologous recombination to occur and for the rescue of recombinant competent adenovirus.
Figure 3.
Figure 3.
Effect of vector design on RCA free production in PER.C6 cell line. In Vector #1 there is a short stretch of homology with the PER.C6 Ad5 sequence that allows homologous recombination. This results in atypical RCA. When the vector has no homology (Vector #2) no homologous recombination occurs and the viral production is RCA free.
Figure 4.
Figure 4.
RCA free vector production in 293-VLI Cells. An ∼8 kb sequence of DNA containing advantageous genes, the spacer, will be incorporated into the HEK293 Ad sequence at nt3510. While this does not prevent homologous recombination due to retention of homology between the genome and Ad vector, packaging of the Ad vector will be suppressed due to the increased size.
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References

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