Scalable and genetically stable recombinant adeno-associated virus (rAAV) production systems combined with facile adaptability for an extended repertoire of AAV serotypes are required to keep pace with the rapidly increasing clinical demand. For scalable high-titer production of the full range of rAAV serotypes 1–12, we developed OneBac, consisting of stable insect Sf9 cell lines harboring silent copies of AAV1–12 rep and cap genes induced upon infection with a single baculovirus that also carries the rAAV genome. rAAV burst sizes reach up to 5×105 benzonase-resistant, highly infectious genomic particles per cell, exceeding typical yields of current rAAV production systems. In contrast to recombinant rep/cap baculovirus strains currently employed for large-scale rAAV production, the Sf9rep/cap cell lines are genetically stable, leading to undiminished rAAV burst sizes over serial passages. Thus, OneBac combines full AAV serotype options with the capacity for stable scale-up production, the current bottleneck for the transition of AAV from gene therapy trials to routine clinical treatment.
Get full access to this article
View all access options for this article.
References
1.
AslanidiG., LambK., and ZolotukhinS. (2009). An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells. Proc. Natl. Acad. Sci. USA, 106, 5059–5064.
2.
BecerraS.P., KoczotF., FabischP., et al. (1988). Synthesis of adeno-associated virus structural proteins requires both alternative mRNA splicing and alternative initiations from a single transcript. J. Virol., 62, 2745–2754.
3.
BergerI., FitzgeraldD.J., and RichmondT.J. (2004). Baculovirus expression system for heterologous multiprotein complexes. Nat. Biotechnol., 22, 1583–1587.
4.
CecchiniS., ViragT., and KotinR.M. (2011). Reproducible high yields of recombinant adeno-associated virus produced using invertebrate cells in 0.02- to 200-liter cultures. Hum. Gene Ther., 22, 1021–1030.
5.
ChadeufG., CironC., MoullierP., et al. (2005). Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery. Mol. Ther., 12, 744–753.
6.
ChenH. (2008). Intron splicing-mediated expression of AAV Rep and Cap genes and production of AAV vectors in insect cells. Mol. Ther., 16, 924–930.
7.
ChenH. (2012). Exploiting the intron-splicing mechanism of insect cells to produce viral vectors harboring toxic genes for suicide gene therapy. Mol. Ther. Nucleic Acids, 1, e57.
8.
ClarkK.R., VoulgaropoulouF., and 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.
9.
ClementN., KnopD.R., and ByrneB.J. (2009). Large-scale adeno-associated viral vector production using a herpesvirus-based system enables manufacturing for clinical studies. Hum. Gene Ther., 20, 796–806.
10.
ConwayJ., RhysC., ZolotukhinI., et al. (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.
11.
ExcoffonK.J., KoerberJ.T., DickeyD.D., et al. (2009). Directed evolution of adeno-associated virus to an infectious respiratory virus. Proc. Natl. Acad. Sci. USA, 106, 3865–3870.
12.
GalibertL., and MertenO.W. (2011). Latest developments in the large-scale production of adeno-associated virus vectors in insect cells toward the treatment of neuromuscular diseases. J. Invertebr. Pathol., 107Suppl, S80–S93.
13.
GaoG.P., AlviraM.R., WangL., et al. (2002). Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc. Natl. Acad. Sci. USA, 99, 11854–11859.
14.
GaoG., AlviraM.R., SomanathanS., et al. (2003). Adeno-associated viruses undergo substantial evolution in primates during natural infections. Proc. Natl. Acad. Sci. USA, 100, 6081–6086.
15.
GrimmD., KernA., RittnerK., et al. (1998). Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum. Gene Ther., 9, 2745–2760.
16.
GrimmD., KayM.A., and KleinschmidtJ.A. (2003). Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6. Mol. Ther., 7, 839–850.
17.
HeilbronnR., EngstlerM., WegerS., et al. (2003). ssDNA-dependent colocalization of adeno-associated virus Rep and herpes simplex virus ICP8 in nuclear replication domains. Nucleic Acids Res., 31, 6206–6213.
18.
HüserD., WegerS., and HeilbronnR. (2003). Packaging of human chromosome 19-specific adeno-associated virus (AAV) integration sites in AAV virions during AAV wild-type and recombinant AAV vector production. J. Virol., 77, 4881–4887.
19.
KangW., WangL., HarrellH., et al. (2008). An efficient rHSV-based complementation system for the production of multiple rAAV vector serotypes. Gene Ther., 16, 229–239.
20.
KasteleinJ.J., RossC.J., and HaydenM.R. (2013). From mutation identification to therapy: discovery and origins of the first approved gene therapy in the Western world. Hum. Gene Ther., 24, 472–478.
21.
KohlbrennerE., AslanidiG., NashK., et al. (2005). Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system. Mol. Ther., 12, 1217–1225.
22.
KuckD., KernA., and KleinschmidtJ.A. (2007). Development of AAV serotype-specific ELISAs using novel monoclonal antibodies. J. Virol. Methods, 140, 17–24.
23.
LockM., AlviraM., VandenbergheL.H., et al. (2010). Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale. Hum. Gene Ther., 21, 1259–1271.
24.
MartinJ., FrederickA., LuoY., et al. (2013). Generation and characterization of adeno-associated virus producer cell lines for research and preclinical vector production. Hum. Gene Ther. Methods, 24, 253–269.
25.
MingozziF., and HighK.A. (2011). Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat. Rev. Genet., 12, 341–355.
26.
MitchellM., NamH.J., CarterA., et al. (2009). Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 9. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 65, 715–718.
27.
MoriS., WangL., TakeuchiT., et al. (2004). Two novel adeno-associated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein. Virology, 330, 375–383.
28.
NathwaniA.C., TuddenhamE.G., RangarajanS., et al. (2011). Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N. Engl. J. Med., 365, 2357–2365.
29.
NonyP., ChadeufG., TessierJ., et al. (2003). Evidence for packaging of rep-cap sequences into adeno-associated virus (AAV) type 2 capsids in the absence of inverted terminal repeats: a model for generation of rep-positive AAV particles. J. Virol., 77, 776–781.
30.
SchmidtM., VoutetakisA., AfioneS., et al. (2008). Adeno-associated virus type 12 (AAV12): a novel AAV serotype with sialic acid- and heparan sulfate proteoglycan-independent transduction activity. J. Virol., 82, 1399–1406.
31.
SmithR.H., LevyJ.R., and KotinR.M. (2009). A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells. Mol. Ther., 17, 1888–1896.
32.
SonntagF., BlekerS., LeuchsB., et al. (2006). Adeno-associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus. J. Virol., 80, 11040–11054.
33.
SonntagF., SchmidtK., and KleinschmidtJ.A. (2010). A viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proc. Natl. Acad. Sci. USA, 107, 10220–10225.
34.
SonntagF., KotherK., SchmidtK., et al. (2011). The assembly-activating protein promotes capsid assembly of different adeno-associated virus serotypes. J. Virol., 85, 12686–12697.
35.
ThomasD.L., WangL., NiamkeJ., et al. (2009). Scalable recombinant adeno-associated virus production using recombinant herpes simplex virus type 1 coinfection of suspension-adapted mammalian cells. Hum. Gene Ther., 20, 861–870.
36.
UrabeM., DingC., and KotinR.M. (2002). Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum. Gene Ther., 13, 1935–1943.
37.
UrabeM., NakakuraT., XinK.Q., et al. (2006). Scalable generation of high-titer recombinant adeno-associated virus type 5 in insect cells. J. Virol., 80, 1874–1885.
38.
ViragT., CecchiniS., and KotinR.M. (2009). Producing recombinant adeno-associated virus in foster cells: overcoming production limitations using a baculovirus-insect cell expression strategy. Hum. Gene Ther., 20, 807–817.
39.
WinterK., von KietzellK., HeilbronnR., et al. (2012). Roles of E4orf6 and VA I RNA in adenovirus-mediated stimulation of human parvovirus B19 DNA replication and structural gene expression. J. Virol., 86, 5099–5109.
40.
WistubaA., WegerS., KernA., et al. (1995). Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins. J. Virol., 69, 5311–5319.
41.
WistubaA., KernA., WegerS., et al. (1997). Subcellular compartmentalization of adeno-associated virus type 2 assembly. J. Virol., 71, 1341–1352.
42.
XiaoX., LiJ., and SamulskiR.J. (1998). Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J. Virol., 72, 2224–2232.
43.
ZolotukhinS. (2005). Production of recombinant adeno-associated virus vectors. Hum. Gene Ther., 16, 551–557.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
