The clinical application of self-inactivating (SIN) retroviral vectors requires an efficient vector production technology. To enable production of γ-retroviral SIN vectors from stable producer cells, new targetable HEK293-based producer clones were selected, providing amphotropic, GALV, or RD114 pseudotyping. Viral vector expression constructs can reliably be inserted at a predefined genomic locus via Flp-recombinase-mediated cassette exchange. Introduction of a clean-up step, mediated by Cre-recombinase, allows the removal of residual sequences that were required for targeting and selection, but were dispensable for the final producer clones and eliminated homology-driven recombination between the tagging and the therapeutic vector. The system was used to establish GALV and RD114 pseudotyping producer cells (HG- and HR820) for a clinically relevant long terminal repeat–driven therapeutic vector, designed for the transfer of a recombinant TCR that delivered titers in the range of 2×107 infectious particles (IP)/ml. Production capacity of the amphotropic producer cell (HA820) was challenged by a therapeutic SIN vector transferring the large COL7A1 cDNA. The final producer clone delivered a titer of 4×106 IP/ml and the vector containing supernatant was used directly to functionally restore primary fibroblasts and keratinocytes isolated from recessive dystrophic epidermolysis bullosa patients. Thus, the combinatorial approach (fc-technology) to generate producer cells for therapeutic γ-retroviral (SIN) vectors is feasible, is highly efficient, and allows their safe production and application in clinical trials.
Get full access to this article
View all access options for this article.
References
1.
AiutiA., SlavinS., AkerM., et al. (2002). Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science, 296, 2410–2413.
2.
BaumC., DullmannJ., LiZ., et al. (2003). Side effects of retroviral gene transfer into hematopoietic stem cells. Blood, 101, 2099–2114.
BraunC.J., BoztugK., ParuzynskiA., et al. (2014). Gene therapy for Wiskott-Aldrich syndrome—long-term efficacy and genotoxicity. Sci. Transl. Med., 6, 227ra33.
5.
BrentjensR.J., DavilaM.L., RiviereI., et al. (2013). CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci. Transl. Med., 5, 177ra38.
6.
CarrondoM., PanetA., WirthD., et al. (2011). Integrated strategy for the production of therapeutic retroviral vectors. Hum. Gene Ther., 22, 370–379.
7.
Cavazzana-CalvoM., Hacein-BeyS., De Saint BasileG., et al. (2000). Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science, 288, 669–672.
8.
CiuffiA., LlanoM., PoeschlaE., et al. (2005). A role for LEDGF/p75 in targeting HIV DNA integration. Nat. Med., 11, 1287–1289.
9.
ConeR.D., and MulliganR.C. (1984). High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range. Proc. Natl. Acad. Sci. USA, 81, 6349–6353.
10.
Corrigan-CurayJ., Cohen-HaguenauerO., O'ReillyM., et al. (2012). Challenges in vector and trial design using retroviral vectors for long-term gene correction in hematopoietic stem cell gene therapy. Mol. Ther., 20, 1084–1094.
11.
EngelsB., CamH., SchulerT., et al. (2003). Retroviral vectors for high-level transgene expression in T lymphocytes. Hum. Gene Ther., 14, 1155–1168.
12.
FehseB., KustikovaO.S., LiZ., et al. (2002). A novel “sort-suicide” fusion gene vector for T cell manipulation. Gene Ther., 9, 1633–1638.
13.
FukushigeS., and SauerB. (1992). Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA, 89, 7905–7909.
14.
GallaM., WillE., KraunusJ., et al. (2004). Retroviral pseudotransduction for targeted cell manipulation. Mol. Cell, 16, 309–315.
15.
Gama-NortonL., HerrmannS., SchuchtR., et al. (2010). Retroviral vector performance in defined chromosomal Loci of modular packaging cell lines. Hum. Gene Ther., 21, 979–991.
16.
GhaniK., CottinS., KamenA., and CarusoM. (2007). Generation of a high-titer packaging cell line for the production of retroviral vectors in suspension and serum-free media. Gene Ther., 14, 1705–1711.
17.
GhaniK., WangX., De Campos-LimaP.O., et al. (2009). Efficient human hematopoietic cell transduction using RD114- and GALV-pseudotyped retroviral vectors produced in suspension and serum-free media. Hum. Gene Ther., 20, 966–974.
18.
GijsbersR., RonenK., VetsS., et al. (2010). LEDGF hybrids efficiently retarget lentiviral integration into heterochromatin. Mol. Ther., 18, 552–560.
19.
Hacein-Bey-AbinaS., Von KalleC., SchmidtM., et al. (2003). LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science, 302, 415–419.
20.
HassanA., BoothC., BrightwellA., et al. (2012). Outcome of hematopoietic stem cell transplantation for adenosine deaminase-deficient severe combined immunodeficiency. Blood, 120, 3615–3624; quiz 3626.
21.
HeemskerkM.H., HoogeboomM., De PausR.A., et al. (2003). Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region. Blood, 102, 3530–3540.
22.
HoltkampS., KreiterS., SelmiA., et al. (2006). Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood, 108, 4009–4017.
23.
LeisegangM., EngelsB., MeyerhuberP., et al. (2008). Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette. J. Mol. Med. (Berl.), 86, 573–583.
24.
LewinskiM.K., YamashitaM., EmermanM., et al. (2006). Retroviral DNA integration: viral and cellular determinants of target-site selection. PLoS Pathog., 2, e60.
25.
LlanoM., SaenzD.T., MeehanA., et al. (2006). An essential role for LEDGF/p75 in HIV integration. Science, 314, 461–464.
26.
LoewR., MeyerY., KuehlckeK., et al. (2010). A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating gamma-retroviral vectors using targeted integration. Gene Ther., 17, 272–280.
27.
LouisC.U., SavoldoB., DottiG., et al. (2011). Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma. Blood, 118, 6050–6056.
28.
LutzR., and BujardH. (1997). Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res., 25, 1203–1210.
29.
MarkowitzD., GoffS., and BankA. (1988). Construction and use of a safe and efficient amphotropic packaging cell line. Virology, 167, 400–406.
30.
MecklenbeckS., ComptonS.H., MejiaJ.E., et al. (2002). A microinjected COL7A1-PAC vector restores synthesis of intact procollagen VII in a dystrophic epidermolysis bullosa keratinocyte cell line. Hum. Gene Ther., 13, 1655–1662.
31.
MillerA.D., and ButtimoreC. (1986). Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol. Cell Biol., 6, 2895–2902.
32.
MillerA.D., LawM.F., and VermaI.M. (1985). Generation of helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene. Mol. Cell Biol., 5, 431–437.
33.
MillerA.D., GarciaJ.V., Von SuhrN., et al. (1991). Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J. Virol., 65, 2220–2224.
34.
ModlichU., and BaumC. (2009). Preventing and exploiting the oncogenic potential of integrating gene vectors. J. Clin. Invest., 119, 755–758.
35.
ModlichU., BohneJ., SchmidtM., et al. (2006). Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity. Blood, 108, 2545–2553.
36.
ModlichU., NavarroS., ZychlinskiD., et al. (2009). Insertional transformation of hematopoietic cells by self-inactivating lentiviral and gammaretroviral vectors. Mol. Ther., 17, 1919–1928.
37.
MontiniE., CesanaD., SchmidtM., et al. (2009). The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy. J. Clin. Invest., 119, 964–975.
38.
OttM.G., SchmidtM., SchwarzwaelderK., et al. (2006). Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat. Med., 12, 401–409.
39.
PietschmannT., HeinkeleinM., HeldmannM., et al. (1999). Foamy virus capsids require the cognate envelope protein for particle export. J. Virol., 73, 2613–2621.
40.
SchlakeT., and BodeJ. (1994). Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry, 33, 12746–12751.
41.
SchuchtR., CoroadinhaA.S., Zanta-BoussifM.A., et al. (2006). A new generation of retroviral producer cells: predictable and stable virus production by Flp-mediated site-specific integration of retroviral vectors. Mol. Ther., 14, 285–292.
42.
SharmaA., LarueR.C., PlumbM.R., et al. (2013). BET proteins promote efficient murine leukemia virus integration at transcription start sites. Proc. Natl. Acad. Sci. USA, 110, 12036–12041.
43.
SilversR.M., SmithJ.A., SchowalterM., et al. (2010). Modification of integration site preferences of an HIV-1-based vector by expression of a novel synthetic protein. Hum. Gene Ther., 21, 337–349.
44.
SommermeyerD., and UckertW. (2010). Minimal amino acid exchange in human TCR constant regions fosters improved function of TCR gene-modified T cells. J. Immunol., 184, 6223–6231.
45.
SoneokaY., CannonP.M., RamsdaleE.E., et al. (1995). A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res., 23, 628–633.
46.
SprangerS., JeremiasI., WildeS., et al. (2012). TCR-transgenic lymphocytes specific for HMMR/Rhamm limit tumor outgrowth in vivo. Blood, 119, 3440–3449.
47.
SteinS., OttM.G., Schultze-StrasserS., et al. (2010). Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat. Med., 16, 198–204.
48.
TiteuxM., PendariesV., Zanta-BoussifM.A., et al. (2010). SIN retroviral vectors expressing COL7A1 under human promoters for ex vivo gene therapy of recessive dystrophic epidermolysis bullosa. Mol. Ther., 18, 1509–1518.
49.
VerhoeyenE., HauserH., and WirthD. (2001). Evaluation of retroviral vector design in defined chromosomal loci by Flp-mediated cassette replacement. Hum. Gene Ther., 12, 933–944.
50.
WeiherH., BarklisE., OstertagW., and JaenischR. (1987). Two distinct sequence elements mediate retroviral gene expression in embryonal carcinoma cells. J. Virol., 61, 2742–2746.
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.
