These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Efficient recombinant adeno-associated virus production by a stable rep-cap HeLa cell line correlates with adenovirus-induced amplification of the integrated rep-cap genome.
    Author: Chadeuf G, Favre D, Tessier J, Provost N, Nony P, Kleinschmidt J, Moullier P, Salvetti A.
    Journal: J Gene Med; 2000; 2(4):260-8. PubMed ID: 10953917.
    Abstract:
    BACKGROUND: A possible procedure for the production of clinical grade recombinant adeno-associated virus type 2 (rAAV) would include the use of packaging cell lines, harboring the rep-cap genes and the vector, combined with a replication defective adenoviral plasmid to provide the helper activities. Several studies have already shown that rAAV can be efficiently assembled by infecting the stable packaging cell line with adenovirus. However, the direct comparison with an adenoviral plasmid has never been reported. METHODS: To investigate this point, a clone of HeLa and 293 cells harboring one to two rep-cap copies per cell genome (HeRC32 and 293RC21, respectively) were generated. Recombinant AAV was produced by transiently transfecting the AAVCMVLacZ vector and supplying the adenoviral helper activities by either wild-type adenovirus or an adenoviral plasmid (pAdc). As a control, rAAV was similarly produced from naive Hela and 293 cells additionally transfected with a rep-cap plasmid. RESULTS: Despite satisfactory rAAV yields from Hela and 293 cells, we show that those from HeRC32 and 293RC21 cells dramatically decrease when adenovirus is replaced by the adenoviral plasmid (pAdc). The analysis performed to identify the factors hampering efficient rAAV assembly by HeRC32 cells in the presence of pAdc shows that: (1) while upon adenovirus infection the integrated rep-cap genome undergoes a dramatic amplification leading to a 100-fold increase in the rep-cap copy number, no amplification is detected upon transfection of pAdc; (2) in pAdc-transfected HeRC32 cells, the intracellular localization of the adenovirus E4orf6 and E1B-55kDa proteins is abnormal as compared to adenovirus-infected cells. CONCLUSIONS: This study documents that stable rep-cap cells lines are severely hampered for rAAV assembly when a replicative adenovirus is substituted with an adenoviral plasmid. Furthermore, our results also suggest that the lack of amplification of the rep-cap genes, eventually combined with the altered distribution of the adenoviral proteins, E4orf6 and E1B-55kDa, is related to the low rAAV yields observed under these conditions.
    [Abstract] [Full Text] [Related] [New Search]