Abstract
The possibility of insertional mutagenesis in retroviral gene therapy can be reduced by using a vector lacking the enhancer sequence present in the U3 of the long-terminal repeats. However, such vectors suffer from many pitfalls. We attempted to improve a murine leukemia virus-based retroviral vector containing the enhancer-free U3, first by making it easier to construct a producer line and then by introducing the cellular RPL10 promoter as an internal promoter. The reverse orientation of the expression cassette of the transgene was found to give higher transducing titer and higher-level gene expression. The deletion analysis revealed that the 54-bp-long sequence of U3 (34 and 20 bp present at 5′ and 3′ extreme ends, respectively) was sufficient for the functions of retroviral vectors. The data from the in vitro cell culture assay indicated that the final construct, ROK, containing all these features, had little cis-activation activity, even if it was placed right upstream from the RNA start site of the neighboring gene. Our data suggested that the newly developed vector might provide increased safety, while still producing high viral titer from a stable producer line and high-level gene expression in various target cells including human CD34+ stem cells.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Edelstein ML, Abedi MR, Wixon J, Edelstein RM . Gene therapy clinical trials worldwide 1989-2004-an overview. J Gene Med 2004; 6: 597–602.
Uren A, Kool J, Berns A, van Lohuizen M . Retroviral insertional mutagenesis: past, present and future. Oncogene 2005; 24: 7656–7672.
Stocking C, Bergholz U, Friel J, Klingler K, Wagener T, Starke C et al. Distinct classes of factor-independent mutants can be isolated after retroviral mutagenesis of a human myeloid stem cell line. Growth Factors 1993; 8: 197–209.
Cavazzana-Calvo M, Hacein-Bey S, Basile G, Gross F, Yvon E, Nusbaum P et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000; 288: 669.
Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack M, Wulffraat N, Leboulch P et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302: 415–419.
Hacein-Bey-Abina S, Garrigue A, Wang G, Soulier J, Lim A, Morillon E et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest 2008; 118: 3132.
Howe SJ, Mansour MR, Schwarzwaelder K, Bartholomae C, Hubank M, Kempski H et al. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J Clin Invest 2008; 118: 3143–3150.
Ott MG, Schmidt M, Schwarzwaelder K, Stein S, Siler U, Koehl U et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 2006; 12: 401–409.
Recchia A, Mavilio F . Site-specific integration into the HUMAN genome: ready for clinical application? Rejuvenation Res 2006; 9: 446–449.
Su K, Wang D, Ye J, Kim YC, Chow SA . Site-specific integration of retroviral DNA in human cells using fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc-finger protein E2C. Methods 2009; 47: 269–276.
Newrzela S, Cornils K, Li Z, Baum C, Brugman MH, Hartmann M et al. Resistance of mature T cells to oncogene transformation. Blood 2008; 112: 2278–2286.
Sugimoto Y, Hrycyna CA, Aksentijevich II, Pastan II, Gottesman MM . Coexpression of a multidrug-resistance gene (MDR1) and herpes simplex virus thymidine kinase gene as part of a bicistronic messenger RNA in a retrovirus vector allows selective killing of MDR1-transduced cells. Clin Cancer Res 1995; 1: 447–457.
Li Z, Dullmann J, Schiedlmeier B, Schmidt M, von Kalle C, Meyer J et al. Murine leukemia induced by retroviral gene marking. Science 2002; 296: 497.
Yu S, Ruden T, Kantoff P, Garber C, Seiberg M, Ruther U et al. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc Natl Acad Sci 1986; 83: 3194–3198.
Yee J, Moores J, Jolly D, Wolff J, Respess J, Friedmann T . Gene expression from transcriptionally disabled retroviral vectors. Proc Natl Acad Sci USA 1987; 84: 5197–5201.
Schambach A, Mueller D, Galla M, Verstegen M, Wagemaker G, Loew R et al. Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors. Gene Ther 2006; 13: 1524–1533.
Boshart M, Weber F, Jahn G, Dorsch-Hasler K, Fleckenstein B, Schaffner W . A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 1985; 41: 521–530.
Moreau-Gachelin F, Ray D, de Both NJ, van der Feltz MJ, Tambourin P, Tavitian A . Spi-1 oncogene activation in Rauscher and Friend murine virus-induced acute erythroleukemias. Leukemia 1990; 4: 20–23.
Prosch S, Stein J, Staak K, Liebenthal C, Volk HD, Kruger DH . Inactivation of the very strong HCMV immediate early promoter by DNA CpG methylation in vitro. Biol Chem Hoppe Seyler 1996; 377: 195–201.
Emerman M, Temin HM . Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell 1984; 39: 449–467.
Moreno-Carranza B, Gentsch M, Stein S, Schambach A, Santilli G, Rudolf E et al. Transgene optimization significantly improves SIN vector titers, gp91phox expression and reconstitution of superoxide production in X-CGD cells. Gene Ther 2009; 16: 111–118.
Hong Y, Yu SS, Kim JM, Lee K, Na YS, Whitley CB et al. Construction of a high efficiency retroviral vector for gene therapy of Hunter's syndrome. J Gene Med 2003; 5: 18–29.
Yu S, Kim J, Kim S . High efficiency retroviral vectors that contain no viral coding sequences. Gene Ther 2000; 7: 794–804.
Potter H, Weir L, Leder P . Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci USA 1984; 81: 7161–7165.
Hong Y, Yu SS, Yoon NK, Kang SJ, Lee JT, Kim S et al. Development of an in vitro cell culture assay system for measuring the activation of a neighbouring gene by the retroviral vector. J Gene Med 2008; 10: 847–854.
Cooper S, Trinklein N, Anton E, Nguyen L, Myers R . Comprehensive analysis of transcriptional promoter structure and function in 1% of the human genome. Genome Res 2006; 16: 1–10.
Soneoka Y, Cannon PM, Ramsdale EE, Griffiths JC, Romano G, Kingsman SM et al. A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res 1995; 23: 628–633.
Zychlinski D, Schambach A, Modlich U, Maetzig T, Meyer J, Grassman E et al. Physiological promoters reduce the genotoxic risk of integrating gene vectors. Mol Ther 2008; 16: 718–725.
Titeux M, Pendaries V, Zanta-Boussif MA, Decha A, Pironon N, Tonasso L et al. SIN retroviral vectors expressing COL7A1 under human promoters for ex vivo gene therapy of recessive dystrophic epidermolysis bullosa. Mol Ther 2010; 18: 1509–1518.
Zhen L, King A, Xiao Y, Chanock S, Orkin S, Dinauer M . Gene targeting of X chromosome-linked chronic granulomatous disease locus in a human myeloid leukemia cell line and rescue by expression of recombinant gp91phox. Proc Natl Acad Sci USA 1993; 90: 9832–9836.
Bandyopadhyay P, Temin H . Expression of complete chicken thymidine kinase gene inserted in a retrovirus vector. Mol Cell Biol 1984; 4: 749–754.
Ishikawa H, Nakata K, Mawatari F, Ueki T, Tsuruta S, Ido A et al. Retrovirus-mediated gene therapy for hepatocellular carcinoma with reversely oriented therapeutic gene expression regulated by α-fetoprotein enhancer/promoter. Biochem Biophys Res Commun 2001; 287: 1034–1040.
Kozak S, Kabat D . Ping-pong amplification of a retroviral vector achieves high-level gene expression: human growth hormone production. J Virol 1990; 64: 3500–3508.
Ramezani A, Hawley TS, Hawley RG . Combinatorial incorporation of enhancer-blocking components of the chicken beta-globin 5′HS4 and human T-cell receptor alpha/delta BEAD-1 insulators in self-inactivating retroviral vectors reduces their genotoxic potential. Stem Cells 2008; 26: 3257–3266.
Gaspar H, Parsley K, Howe S, King D, Gilmour K, Sinclair J et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet 2004; 364: 2181–2187.
Hacein-Bey-Abina S, Le Deist F, Carlier F, Bouneaud C, Hue C, De Villartay JP et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 2002; 346: 1185–1193.
Kim S, Lee K, Kim M, Kang S, Joo C, Kim J et al. Factors affecting the performance of different long terminal repeats in the retroviral vector. Biochem Biophys Res Commun 2006; 343: 1017–1022.
Naumann N, De Ravin S, Choi U, Moayeri M, Whiting-Theobald N, Linton G et al. Simian immunodeficiency virus lentivector corrects human X-linked chronic granulomatous disease in the NOD/SCID mouse xenograft. Gene Ther 2007; 14: 1513–1524.
Kaneko K, Kobayashi H, Onodera O, Miyatake T, Tsuji S . Genomic organization of a cDNA (QM) demonstrating an altered mRNA level in nontumorigenic Wilms’ microcell hybrid cells and its localization to Xq28. Hum Mol Genet 1992; 1: 529–533.
Loew R, Meyer Y, Kuehlcke K, Gama-Norton L, Wirth D, Hauser H et al. A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating gamma-retroviral vectors using targeted integration. Gene Ther 2009; 17: 272–280.
Yu S, Han E, Hong Y, Lee J, Kim S . Construction of a retroviral vector production system with the minimum possibility of a homologous recombination. Gene Ther 2003; 10: 706–711.
Hong Y, Lee K, Yu SS, Kim S, Kim JG, Shin HY . Factors affecting retrovirus-mediated gene transfer to human CD34+ cells. J Gene Med 2004; 6: 724–733.
Gorman C, Moffat L, Howard B . Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 1982; 2: 1044.
Acknowledgements
We would like to thank Kyungmi Koh, Jonghyun Choi, Hwajin Kong, Hyelim Cho and Hae-Sook Ahn for their assistance in cloning various cellular promoters and mutant forms of 3′LTR. This work was supported in part by the grant given to S Kim's university laboratory by Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology (#2010K000829), in which ViroMed Co., Ltd. is a participating company.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
Sunyoung Kim, Sujeong Kim and Ja Young Kim are employees of ViroMed Co., Ltd.
Additional information
Supplementary Information accompanies the paper on Gene Therapy website
Supplementary information
Rights and permissions
About this article
Cite this article
Jang, J., Lee, JT., Lee, K. et al. Development of murine leukemia virus-based retroviral vectors with a minimum possibility of cis-activation. Gene Ther 18, 240–249 (2011). https://doi.org/10.1038/gt.2010.135
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/gt.2010.135