Biomedical Engineering Reference
In-Depth Information
26.
Hum Gene Ther
. 2009
Aug;20(8):861-70. Scalable
recombinant adeno-associated
virus production using
recombinant herpes simplex
virus type 1 coinfection of
suspension-adapted mammalian
cells. Thomas DL, Wang L,
Niamke J, Liu J, Kang W, Scotti
MM, Ye GJ, Veres G, Knop DR.
Applied Genetic Technologies
Corporation, Alachua, FL 32615.
Recombinant adeno-associated virus (rAAV)
production systems capable of meeting clinical or
anticipated commercial-scale manufacturing needs
have received relatively little scrutiny compared with
the intense research activity afforded the in vivo and
in vitro evaluation of rAAV for gene transfer.
Previously, we have reported a highly efficient
recombinant herpes simplex virus type 1 (rHSV)
complementation system for rAAV production in
multiple adherent cell lines; however, production in a
scalable format was not demonstrated. Here, we
report rAAV production by rHSV coinfection of baby
hamster kidney (BHK) cells grown in suspension
(sBHK cells), using two ICP27-deficient rHSV
vectors, one harboring a transgene flanked by the
AAV2 inverted terminal repeats and a second bearing
the AAV rep2 and capX genes (where X is any rAAV
serotype). The rHSV coinfection of sBHK cells
produced similar rAAV1/AAT-specific yields (85,400
DNase-resistant particles [DRP]/cell) compared with
coinfection of adherent HEK-293 cells (74,600 DRP/
cell); however, sBHK cells permitted a threefold
reduction in the rHSV-rep2/capX vector multiplicity
of infection, grew faster than HEK-293 cells, retained
specific yields (DRP/cell) at higher cell densities, and
had a decreased virus production cycle. Furthermore,
sBHK cells were able to produce AAV serotypes 1, 2,
5, and 8 at similar specific yields, using multiple
therapeutic genes. rAAV1/AAT production in sBHK
cells was scaled to 10 L disposable bioreactors, using
optimized spinner flask infection conditions, and
resulted in average volumetric productivities as high
as 2.4 × 10(14) DRP/liter.
