A Single Short Stretch of Homology Between Adenoviral Vector and Packaging Cell Line Can Give Rise to Cytopathic Effect-Inducing, Helper-Dependent E1-Positive Particles

Abstract

An undesirable byproduct from recombinant adenoviral vectors is the emergence of replication competent adenovirus (RCA) that result from rare homologous recombination events between the viral E1-containing (permissive) mammalian host cell genome and the virus itself, restoring the E1 gene to the viral genome. To reduce or eliminate the problem of RCA, we evaluated production of a first generation Ad5 vector (Ad5FGF4) in the cell line PER.C6. This E1-transformed human cell line contains only Ad5 nucleotides 459-3510, which precludes double crossover-type homologous recombination because the Ad5FGF-4 only contains 5' Ad5 sequence up to nucleotide 453. The Ad5FGF4 vector does, however, retain 177 nucleotides of the 3' end of the E1B-55K gene that are also present in PER.C6. With only this single region of homology between vector and cell line, we were surprised to detect virus-specific cytopathic effects (CPE) in our cell-based assay for RCA. This CPE-inducing agent was amplified in nonpermissive A549 cells but also supported amplification of the parental Ad5FGF-4. Because we were unable to isolate the CPE-inducing agent in pure form we first identified it as atypical RCA. Polymerase chain reaction (PCR) and Southern blot experiments identified viral DNA segments in which recombination had occurred between the 177 nucleotides of E1B present in both Ad5FGF-4 and PER.C6. The atypical RCA genomes contain a copy of the original (PGK promoter-E1 gene carrying) plasmid used in the construction of the PER.C6 cell line and they retained the parental FGF-4 transgene. However, significant deletions occurred within the recombined genomes in compensation for the large insertion from PER.C6 sequences and resulted in the loss of essential viral genes. This deletion renders these recombinant viruses replication defective, requiring helper functions from remaining parental Ad5FGF-4 for amplification. These atypical RCA entities may be more properly designated as helper-dependent E1-positive particles (HDEPs). This finding shows the importance of avoiding the use of "nonmatched" vectors where any overlap exists between the recombinant vector and E1 sequences in the packaging cell line. The cloning of the FGF-4 transgene into an adenoviral vector specifically "matched" for PER.C6 (lacking the 177 nucleotide region of homology) has allowed extensive virus propagation (Ad5.1FGF-4) with no CPE- or HDEP-like events yet detected.

Common Structure of Rare Replication-Deficient E1-Positive Particles in Adenoviral Vector Batches

“…Many recombinant Ad batches have been produced using the PER.C6 cell line, and RCA have not been detected. We have previously demonstrated the importance of using the appropriate vector-plasmid system in combination with PER.C6 cells, showing that a 177-bp sequence homology at one end of the E1 region is sufficient for recombination between cells and vector (14). In that study we showed that the single crossover event observed gave rise to a helper-dependent, replication-incompetent virus with a low frequency (less than 1 in 10 11 particles).…”

“…This DNA was cotransfected with PacI-digested cosmid pWE/ AdAfIII-rITR (6) into PER.C6 cells (3) using Lipofectamine and the protocol provided by the manufacturer (Life Technologies). Virus propagation and purification have been described previously (14). For each production lot, a separate aliquot from a master virus bank was used for the initial infection.…”

“…A summary of this bioassay has been presented previously (14). In brief, Ad5.1FGF-4 virus samples were used to infect adherent cultures of HeLa cells at 2,000 virus particles/cell.…”

“…This resulted in a cytopathic effect (CPE)-inducing agent that was completely dependent on the presence of the parental recombinant virus for replication. Since the CPE-inducing agent could not be propagated in the absence of recombinant vector, the term helper-dependent-E1-containing particle, or HDEP, was coined (14). We reported that the emergence of both RCA and these HDEPs could be circumvented, using the tailored vector that was specifically adapted for use with PER.C6 cells, after producing 20 production lots in which no CPE-inducing events could be detected.…”

Viral vectors for in vivo gene transfer in Parkinson's disease: Properties and clinical grade production

“…Wild-type virus may contaminate vector preparations, and RCV may be formed during production due to recombination between the vector and the helper sequences found in complementary cell lines or plasmids (Escarpe et al, 2003;Murakami et al, 2002;Nony et al, 2003;Sastry et al, 2005) The presence of wild-type virus or RCV may affect vector expression and mobilization.…”