Differential amplification of adenovirus vectors by flanking the packaging signal with attB/attP-PhiC31 sequences: implications for helper-dependent adenovirus production.
Current strategies to amplify helper-dependent adenovirus, based on excision of the packaging signal, do not routinely reduce helper adenovirus contamination below 1%. Here, we have tested if reducing the efficiency of the packaging process of the helper adenovirus could impair its packaging without affecting helper-dependent adenovirus production. Interestingly, insertion of attB/attP-PhiC31 sequences flanking the packaging signal significantly lengthens adenovirus cycle up to 60 h without reducing virus viability or production yield. This delay occurs in the absence of PhiC31 recombinase indicating that other mechanisms different from excision of packaging signal must be involved. In addition, at 36 h post-coinfection helper-dependent adenovirus are efficiently produced, while production levels of helper attB/attP-modified adenovirus are 100-1000 times lower than controls. Therefore, these results suggest that attB/attP-mediated packaging impairment of the adenovirus genome is an attractive strategy to significantly reduce helper adenovirus contamination in helper-dependent adenovirus preparations, which in turn would facilitate scaling-up processes for clinical grade preparations.
Alba
Raul
R
Center of Animal Biotechnology and Gene Therapy (CBATEG), and Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain.
Hearing
Patrick
P
Bosch
Assumpció
A
Chillon
Miguel
M
D000260:Adenoviruses, Human / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D001287:Attachment Sites, Microbiological / Q000235:genetics*; D002460:Cell Line; D004279:DNA, Viral / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006378:Helper Viruses / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D021382:Protein Sorting Signals / Q000235:genetics*; D045522:Recombinases / Q000235:genetics*; D019065:Virus Assembly
D004279:DNA, Viral; D021382:Protein Sorting Signals; D045522:Recombinases