Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2–secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.
Yla-HerttualaS. Endgame: glybera finally recommended for approval as the first gene therapy drug in the European union. Mol Ther, 2012; 20:1831–1832.
3.
BuningH. Gene therapy enters the pharma market: the short story of a long journey. EMBO Mol Med, 2013; 5:1–3.
4.
KasteleinJJ, RossCJ, HaydenMR. From mutation identification to therapy: discovery and origins of the first approved gene therapy in the western world. Hum Gene Ther, 2013; 24:472–478.
5.
van der LooJC, WrightJF. Progress and challenges in viral vector manufacturing. Hum Mol Genet, 2016; 25:R42–52.
6.
AyusoE, MingozziF, BoschF. Production, purification and characterization of adeno-associated vectors. Curr Gene Ther, 2010; 10:423–436.
7.
ClementN, GriegerJC. Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol Ther Methods Clin Dev, 2016; 3:1–7.
8.
MaguireAM, SimonelliF, PierceEA, et al.Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med, 2008; 358:2240–2248.
9.
AllayJA, SleepS, LongS, et al.Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial. Hum Gene Ther, 2011; 22:595–604.
10.
FlotteTR, TrapnellBC, HumphriesM, et al.Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing α1-antitrypsin: interim results. Hum Gene Ther. 2011; 22:1239–1247.
11.
ScariaA, LeHalpereA, PurvisA, et al.Preliminary results of a phase 1, open-label, safety and tolerability study of a single intravitreal injection of AAV2-sFLT01 in patients with neovascular age-related macular degeneration. Mol Ther, 2016; 24(supp1):pS98.
12.
ClarkKR, VoulgaropoulouF, FraleyDM, JohnsonPR. Cell lines for the production of recombinant adeno-associated virus. Hum Gene Ther, 1995; 6:1329–1341.
GreenbergG, YaroshinskyA, ZseboKM, et al.Design of a phase 2b trial of intracoronary administration of AAV1/SERCA2a in patients with advanced heart failure: the CUPID 2 trial (calcium up-regulation by percutaneous administration of gene therapy in cardiac disease phase 2b. JACC Heart Fail, 2014; 2:84–92.
15.
MartinJ, FrederickA, LuoY, et al.Generation and characterization of adeno-associated virus producer cell lines for research and preclinical vector production. Hum Gene Ther Methods, 2013; 24:253–269.
16.
SuroskyRT, UrabeM, GodwinSG, et al.Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome. J Virol, 1997; 71:7951–7959.
17.
PieroniL, FipaldiniC, MonciottiA, et al.Targeted integration of adeno-associated virus-derived plasmids in transfected human cells. Virology, 1998; 249:249–259.
18.
TsunodaH, HayakawaT, SakuragawaN, et al.Site-specific integration of adeno-associated virus-based plasmid vectors in lipofected HeLa cells. Virology, 2000; 268:391–401.
19.
GaoGP, QuG, FaustLZ, et al.High-titer adeno-associated viral vectors from a rep/cap cell line and hybrid shuttle virus. Hum Gene Ther, 1998; 9:2353–2362.
20.
GaoGP, LuF, SanmiguelJC, et al.Rep/cap gene amplification and high-yield production of AAV in an A549 cell line expressing Rep/Cap. Mol Ther, 2002; 5:644–649.
21.
QiaoC, WangB, ZhuX, et al.A novel gene expression control system and its use in stable, high-titer 293 cell-based adeno-associated virus packaging cell lines. J Virol, 2002; 76:13015–13027.
22.
FarsonD, HardingTC, TaoL, et al.Development and characterization of a cell line for large-scale, serum-free production of recombinant adeno-associated viral vectors. J Gene Med, 2004; 6:1369–1381.
23.
LiuXL, VoulgaropoulouF, ChenR, et al.Selective rep-cap gene amplification as a mechanism for high-titer recombinant AAV production from stable cell lines. Mol Ther, 2000; 2:394–403.
24.
KotinRM, SiniscalcoM, SamulskiRJ, et al.Site-specific integration by adeno-associated virus. Proc Natl Acad Sci, 1990; 87:2211–2215.
25.
SamulskiRJ, ZhuX, XiaoX, et al.Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J, 1991; 10:3941–3950.
26.
YoungSM, SamulskiRJ. Adeno-associated virus (AAV) site-specific recombination does not require a Rep-dependent origin of replication within the AAV terminal repeat. Proc Natl Acad Sci, 2001; 98:13525–13530.
27.
PhilpottNJ, Giraud-WaliC, DupuisC, et al.Efficient integration of recombinant adeno-associated virus DNA vectors requires a p5-rep sequence in cis. J Virol, 2002; 76:5411–5421.
28.
PhilpottNJ, GomosJ, BernsKI, Falck-PedersenE. A p5 integration efficiency element mediates Rep-dependent integration into AAVS1 at chromosome 19. Proc Natl Acad Sci U S A, 2002; 99:12381–12385.
29.
LindenRM, WinocourE, BernsKI. The recombination signals for adeno-associated virus site-specific integration. Proc Natl Acad Sci U S A, 1996; 93:7966–7972.
30.
LindenRM, WardP, GiraudC, et al.Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A, 1996; 93:11288–11294.
31.
MenesesP, BernsKI, WinocourE. DNA sequence motifs which direct adeno-associated virus site-specific integration in a model system. J Virol, 2000; 74:6213–6216.
32.
MusayevFN, Zarate-PerezF, BishopC, et al.Structural insights into the assembly of the adeno-associated virus type 2 Rep68 protein on the integration site AAVS1. J Biol Chem, 2015; 290:27487–27499.
33.
WeitzmanMD, KyostioSRM, KotinRM, OwensRA. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA. Proc Natl Acad Sci U S A, 1994; 91:5808–5812.
34.
HuserD, Gogol-DoringA, LutterT, et al.Integration preferences of wildtype AAV-2 for consensus Rep-binding sites at numerous loci in the human genome. PLoS pathogens, 2010; 6:e1000985.
35.
JanovitzT, KleinIA, OliveiraT, et al.High-throughput sequencing reveals principles of adeno-associated virus serotype 2 integration. J Virol, 2013; 87:8559–8568.
36.
HuserD, Gogol-DoringA, ChenW, HeilbronnR. Adeno-associated virus type 2 wild-type and vector-mediated genomic integration profiles of human diploid fibroblasts analyzed by third-generation PacBio DNA sequencing. J Virol, 2014; 88:11253–11263.
37.
JanovitzT, SadelainM, Falck-Pedersen. Adeno-associated virus type 2 preferentially integrates single genome copies with defined breakpoints. Virol J, 2014; 11:15–20.
38.
PetriK, GabrielR, AgundezL, et al.Presence of a trs-like motif promotes Rep-mediated wild-type adeno-associated virus type 2 integration. J Virol, 2015; 89:7428–7432.
39.
TanI, NgCH, LimL, LeungT. Phosphorylation of a novel myosin binding subunit of protein phosphatase 1 reveals a conserved mechanism in the regulation of actin cytoskeleton. J Biol Chem, 2001; 276:21209–21216.
40.
SmithJR, MaguireS, DavisLA, et al.Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration. Stem Cells, 2008; 26:496–504.
41.
LiuX, PingH, ZhangC. Rapid establishment of a HEK cell line expressing FVIII-BDD using AAV site-specific integration plasmids. BMC Res Notes, 2014; 7:626–631.
42.
HockemeyerD, SoldnerF, BeardC, et al.Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol, 2009; 27:851–857.
43.
ZouJ, SweeneyCL, ChouBK, et al.Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting. Blood, 2011; 117:5561–5572.
44.
DeRavinSS, ReikA, LiuPQ, et al.Targeted gene addition in human CD34+ hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat Biotechnol, 2016; 34:424–429.
45.
SivalingamJ, KenanovD, HanH, et al.Multidimensional genome-wide analyses show accurate FVIII integration by ZFN in primary human cells. Mol Ther, 2016; 24:607–619.
46.
Oceguera-YanezF, KimSI, MatsumotoT, et al.Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives. Methods, 2015; 101:43–55.
47.
DreyerAK, HoffmannD, LachmannN, et al.TALEN-mediated functional correction of X-linked chronic granulomatous disease in patient-derived induced pluripotent stem cells. Biomaterials, 2015; 69:191–200.
48.
GrahamFL, PrevecL. Manipulation of adenovirus vectors. In: MurrayEJ, ed. Gene Transfer and Protocol, vol 7. Clifton, NJ: Humana Press, 1991:109–128.
49.
WardP, WalshCE. Targeted integration of a rAAV vector into the AAVS1 region. Virology, 2012; 433:356–366.
50.
MacvilleM, SchrockE, Padilla-NashH, et al.Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping. Cancer Res, 1999; 59:141–150.
51.
PhilpottNJ, GomosJ, Falck-PedersenE. Transgene expression after rep-mediated site-specific integration into chromosome 19. Hum Gene Ther, 2004; 15:47–61.
52.
MurphyM, Gomos-KlienJ, StankicM, Falck-PedersenE. Adeno-associated virus type 2 p5 promoter: a rep-regulated DNA switch element functioning in transcription, replication and site-specific integration. J Virol, 2007; 81:3721–3730.
53.
GuilbaudM, ChadeufG, AvolioF, et al.Relative influence of the adeno-associated virus (AAV) type 2 p5 element for recombinant AAV vector site-specific integration. J Virol, 2008; 82:2590–2593.
54.
LamartinaS, RoscilliG, RinaudoD, et al.Lipofection of purified adeno-associated virus Rep68 protein: toward a chromosome-targeting nonviral particle. J Virol, 1998; 72:7653–7658.
55.
BalagueC, KallaM, ZhangW. Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome. J Virol, 1997; 71:3299–3306.
56.
KogureD, UrabeM, MizukamiH, et al.Targeted integration of foreign DNA into a defined locus on chromosome 19 in K562 cells using AAV-derived components. Int J Hematol, 2001; 73:469–475.
57.
BeatonA, PalumboP, BernsKI. Expression from the adeno-associated virus p5 and p19 promoters is negatively regulated in trans by the rep protein. J Virol, 1989; 63:4450–4454.
58.
KyostioSR, WonderlingRS, OwensRA. Negative regulation of the adeno-associated virus (AAV) p5 promoter involves both the p5 rep binding site and the consensus ATP-binding motif of the AAV Rep68 protein. J Virol, 1995; 69:6787–6796.
59.
YoungSM, McCartyDM, DegtyarevaN, SamulskiRJ. Roles of adeno-associated virus rep protein and human chromosome 19 in site-specific recombination. J Virol, 2000; 74:3953–3966.
60.
HuttnerNA, GirodA, SchnittgerS, et al.Analysis of site-specific transgene integration following cotransduction with recombinant adeno-associated virus and a Rep encoding plasmid. J Gene Med, 2003; 5:120–129.
61.
HuserD, WegerS, HeilbronnR. Kinetics and frequency of adeno-associated virus site-specific integration into human chromosome 19 monitored by quantitative real-time PCR. J Virol, 2002; 76:7554–7559.
62.
HuserD, WegerS, HeilbronnR. 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, 2003; 77:4881–4887.
63.
YanZ, ZakR, ZhangY, EngelhardtJF. Inverted terminal repeat sequences are important for intermolecular recombination and circularization of adeno-associated virus genomes. J Virol, 2005; 79:364–379.
HolscherC, HorerM, KleinschmidtJA, et al.Cell lines inducibly expressing the adeno-associated virus (AAV) rep gene: requirements for productive replication of rep-negative AAV mutants. J Virol, 1994; 68:7169–7177.
66.
YangQ, ChenF, TrempeJP. Characterization of cell lines that inducibly express the adeno-associated virus Rep proteins. J Virol, 1994; 68:4847–4856.
67.
HenckaertsE, DutheilN, ZeltnerN, et al.Site-specific integration of adeno-associated virus involves partial duplication of the target locus. Proc Natl Acad Sci U S A, 2009; 106:7571–7576.
68.
CheungAK, HogganMD, HauswirthWW, BernsKI. Integration of the adeno-associated virus genome into cellular DNA in latently infected human Detroit 6 cells. J Virol, 1980; 33:739–748.
69.
YangCC, XiaoX, ZhuX, et al.Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro. J Virol, 1997; 71:9231–9247.
70.
YangJ, ZhouW, ZhangY, et al.Concatemerization of adeno-associated virus circular genomes occurs through intermolecular recombination. J Virol, 1999; 73:9468–9477.
71.
HuserD, HeilbronnR. Adeno-associated virus integrates site-specifically into human chromosome 19 in either orientation and with equal kinetics and frequency. J Gen Virol, 2003; 84:133–137.
MillerDG, PetekLM, RussellDW. Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks. Mol Cell Biol, 2003; 23:3550–3557.
PorteusMH, CathomenT, WeitzmanMD, BaltimoreD. Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks. Mol Cell Biol, 2003; 23:3558–3565.
76.
CataldiMP, McCartyDM. Differential effects of DNA double-strand break repair pathways on single-strand and self-complementary adeno-associated virus vector genomes. J Virol, 84:8673–8682.
77.
AdeyA, BurtonJN, KitzmanJO, et al.The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line. Nature, 2013; 500:207–211.
78.
LamartinaS, SporenoE, FattoriE, et al.Characteristics of the adeno-associated virus preintegration site in human chromosome 19: open chromatin conformation and transcription-competent environment. J Virol, 2000; 74:7671–7677.
79.
NambiarB, Sookdeo-CornellC, BertheletteP, et al.Characteristics of minimally oversized adeno-associated virus vectors encoding human factor VIII generated using producer cell lines and triple transfection. Hum Gene Ther Methods, 2017; 28:23–38.
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