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 3-fold 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-liter disposable bioreactors, using optimized spinner flask infection conditions, and resulted in average volumetric productivities as high as 2.4 × 1014 DRP/liter.
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References
1.
Alazard-DanyN., NicolasA., PloquinA., StrasserR., GrecoA., EpsteinA.L., FraefelC., SalvettiA.2009. Definition of herpes simplex virus type 1 helper activities for adeno-associated virus early replication events. PLoS Pathog., 5:e1000340.
2.
AlbuE., HolmesK.V.1973. Isolation and preliminary characterization of the RNA-containing R-type, virus-like particle of BHK-21 cells. J. Virol., 12:1164–1172.
3.
AucoinM.G., PerrierM., KamenA.A.2008. Critical assessment of current adeno-associated viral vector production and quantification methods. Biotechnol. Adv., 26:73–88.
4.
BergmannD.G., WolffD.A.1981. Production of R-type virus-like particles in hamster cells. Intervirology, 16:61–70.
5.
BlochJ., Bachoud-LeviA.C., DeglonN., LefaucheurJ.P., WinkelL., PalfiS., NguyenJ.P., BourdetC., GauraV., RemyP., BrugieresP., BoisseM.F., BaudicS., CesaroP., HantrayeP., AebischerP., PeschanskiM.2004. Neuroprotective gene therapy for Huntington's disease, using polymer-encapsulated cells engineered to secrete human ciliary neurotrophic factor: Results of a phase I study. Hum. Gene Ther., 15:968–975.
6.
BoedekerB.G.2001. Production processes of licensed recombinant factor VIII preparations. Semin. Thromb. Hemost., 27:385–394.
7.
BoothM.J., MistryA., LiX., ThrasherA., CoffinR.S.2004. Transfection-free and scalable recombinant AAV vector production using HSV/AAV hybrids. Gene Ther., 11:829–837.
8.
BullerR.M., JanikJ.E., SebringE.D., RoseJ.A.1981. Herpes simplex virus types 1 and 2 completely help adenovirus-associated virus replication. J. Virol., 40:241–247.
9.
BuningH., PeraboL., CoutelleO., Quadt-HummeS., HallekM.2008. Recent developments in adeno-associated virus vector technology. J. Gene Med., 10:717–733.
10.
CecchiniS., NegreteA., KotinR.M.2008. Toward exascale production of recombinant adeno-associated virus for gene transfer applications. Gene Ther., 15:823–830.
11.
ChristieA., ButlerM.1999. The adaptation of BHK cells to a non-ammoniagenic glutamate-based culture medium. Biotechnol. Bioeng., 64:298–309.
12.
ClarkK.R., VoulgaropoulouF., FraleyD.M., JohnsonP.R.1995. Cell lines for the production of recombinant adeno-associated virus. Hum. Gene Ther., 6:1329–1341.
13.
ClarkK.R., VoulgaropoulouF., JohnsonP.R.1996. A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors. Gene Ther., 3:1124–1132.
14.
ConwayJ.E., ZolotukhinS., MuzyczkaN., HaywardG.S., ByrneB.J.1997. Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap. J. Virol., 71:8780–8789.
15.
ConwayJ.E., RhysC.M., ZolotukhinI., ZolotukhinS., MuzyczkaN., HaywardG.S., ByrneB.J.1999. High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap. Gene Ther., 6:986–993.
16.
Di PasqualeG., DavidsonB.L., SteinC.S., MartinsI., ScudieroD., MonksA., ChioriniJ.A.2003. Identification of PDGFR as a receptor for AAV-5 transduction. Nat. Med., 9:1306–1312.
17.
DurocherY., PhamP.L., St-LaurentG., JacobD., CassB., ChahalP., LauC.J., NalbantogluJ., KamenA.2007. Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells. J. Virol. Methods, 144:32–40.
18.
FarsonD., HardingT.C., TaoL., LiuJ., PowellS., VimalV., YendluriS., KoprivnikarK., HoK., TwittyC., HusakP., LinA., SnyderR.O., DonahueB.A.2004. Development and characterization of a cell line for large-scale, serum-free production of recombinant adeno-associated viral vectors. J. Gene Med., 6:1369–1381.
19.
FengL., GuoM., ZhangS., ChuJ., ZhuangY.2007. Optimization of transfection mediated by calcium phosphate for plasmid rAAV-LacZ (recombinant adeno-associated virus-β-galactosidase reporter gene) production in suspension-cultured HEK-293 (human embryonic kidney 293) cells. Biotechnol. Appl. Biochem., 46:127–135.
20.
FeudnerE., De AlwisM., ThrasherA.J., AliR.R., FauserS.2001. Optimization of recombinant adeno-associated virus production using an herpes simplex virus amplicon system. J. Virol. Methods, 96:97–105.
21.
HermonatP.L., MuzyczkaN.1984. Use of adeno-associated virus as a mammalian DNA cloning vector: Transduction of neomycin resistance into mammalian tissue culture cells. Proc. Natl. Acad. Sci. U.S.A., 81:6466–6470.
22.
HildingerM., BaldiL., StettlerM., WurmF.M.2007. High-titer, serum-free production of adeno-associated virus vectors by polyethyleneimine-mediated plasmid transfection in mammalian suspension cells. Biotechnol. Lett., 29:1713–1721.
23.
KangW., WangL., HarrellH., LiuJ., ThomasD.L., MayfieldT.L., ScottiM.M., YeG.J., VeresG., KnopD.R.2009. An efficient rHSV-based complementation system for the production of multiple rAAV vector serotypes. Gene Ther., 16:229–239.
24.
KnopD.R., HarrellH.2007. Bioreactor production of recombinant herpes simplex virus vectors. Biotechnol. Prog., 23:715–721.
25.
KohlbrennerE., AslanidiG., NashK., ShklyaevS., Campbell-ThompsonM., ByrneB.J., SnyderR.O., MuzyczkaN., WarringtonK.H.Jr., ZolotukhinS.2005. Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system. Mol. Ther., 12:1217–1225.
26.
MenaJ.A., RamirezO.T., PalomaresL.A.2007. Population kinetics during simultaneous infection of insect cells with two different recombinant baculoviruses for the production of rotavirus-like particles. BMC Biotechnol., 7:39.
27.
MistryA.R., De AlwisM., FeudnerE., AliR.R., ThrasherA.J.2002. High-titer stocks of adeno-associated virus from replicating amplicons and herpes vectors. Methods Mol. Med., 69:445–460.
28.
MuzyczkaN., BernsK.I.KnipeD.M., HowleyP.M.2001. Parvoviridae: The viruses and their replication. Fields Virology. Lippincott, Williams & Wilkins: New York, 2327–2360.
29.
NegreteA., YangL.C., MendezA.F., LevyJ.R., KotinR.M.2007. Economized large-scale production of high yield of rAAV for gene therapy applications exploiting baculovirus expression system. J. Gene Med., 9:938–948.
30.
ParkJ.Y., LimB.P., LeeK., KimY.G., JoE.C.2006. Scalable production of adeno-associated virus type 2 vectors via suspension transfection. Biotechnol. Bioeng., 94:416–430.
31.
RiceS.A., KnipeD.M.1990. Genetic evidence for two distinct transactivation functions of the herpes simplex virus α protein ICP27. J. Virol., 64:1704–1715.
ToublancE., BenraissA., BonninD., BlouinV., BrumentN., CartierN., EpsteinA.L., MoullierP., SalvettiA.2004. Identification of a replication-defective herpes simplex virus for recombinant adeno-associated virus type 2 (rAAV2) particle assembly using stable producer cell lines. J. Gene Med., 6:555–564.
34.
TratschinJ.D., WestM.H., SandbankT., CarterB.J.1984. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase. Mol. Cell. Biol., 4:2072–2081.
35.
UrabeM., DingC., KotinR.M.2002. Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum. Gene Ther., 13:1935–1943.
36.
WeindlerF.W., HeilbronnR.1991. A subset of herpes simplex virus replication genes provides helper functions for productive adeno-associated virus replication. J. Virol., 65:2476–2483.
37.
WustnerJ.T., ArnoldS., LockM., RichardsonJ.C., HimesV.B., KurtzmanG., PelusoR.W.2002. Production of recombinant adeno-associated type 5 (rAAV5) vectors using recombinant herpes simplex viruses containing rep and cap. Mol. Ther., 6:510–518.
38.
YeG.-J., DrogemullerC., ThomasD.L., ScottiM.M., KangW., KnopD.R., BoyeS., PechanP., PetersonJ., HauswirthW., ScariaA., WadsworthS.2006. A novel herpes simplex virus helper based production of adeno-associated virus vectors for treatment of retinal angiogenesis. Mol. Ther., 13:S194.
39.
ZhangX., De AlwisM., HartS.L., FitzkeF.W., InglisS.C., BoursnellM.E., LevinskyR.J., KinnonC., AliR.R., ThrasherA.J.1999. High-titer recombinant adeno-associated virus production from replicating amplicons and herpes vectors deleted for glycoprotein H. Hum. Gene Ther., 10:2527–2537.
