RESEARCH ARTICLE
Ef®cient recombinant adeno-associated virus
production by a stable rep-cap HeLa cell line
correlates with adenovirus-induced ampli®cation
of the integrated rep-cap genome
Gilliane Chadeuf
1
David Favre
1
Jacques Tessier
1
Nathalie Provost
1
Pascale Nony
1
Ju
È
rgen Kleinschmidt
2
Philippe Moullier
1
*
Anna Salvetti
1
1
Laboratoire de The
Â
rapie Ge
Â
nique,
CHU Hotel-DIEU, 44035 Nantes
CEDEX 01, France
2
Deutsches Krebsforschungszentrum,
Forschungsschwerpunkt Angewandte
Tumorvirologie, D69120 Heidelberg,
Germany
*Correspondence to: P. Moullier,
Laboratoire de The
Â
rapie Ge
Â
nique,
CHU Hotel-Dieu, Ba
Ã
t. Jean Monnet,
30 Bd Jean Monnet, 44035 Nantes
Cedex 01, France.
E-mail:
moullier@sante.univ-nantes.fr
Received: 10 January 2000
Revised: 22 March 2000
Accepted: 30 March 2000
Published online: 3 April 2000
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 ef®ciently 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 ef®cient 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 ampli®cation
leading to a 100-fold increase in the rep-cap copy number, no ampli®cation 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 ampli®cation 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. Copyright #
2000 John Wiley & Sons, Ltd.
Keywords rAAV; packaging cell lines
Introduction
The ability of recombinant AAV vectors (rAAV) to transduce tissues in vivo
leading to stable gene expression with little or no pathology has contributed
to the widespread use and development of these vectors. More recently, two
THE JOURNAL OF GENE MEDICINE
J Gene Med 2000; 2: 260±268.
Copyright # 2000 John Wiley & Sons, Ltd.