Pharmaceutical Research

, Volume 26, Issue 6, pp 1432–1445 | Cite as

Cell Type-Specific Targeting with Surface-Engineered Lentiviral Vectors Co-displaying OKT3 Antibody and Fusogenic Molecule

  • Haiguang Yang
  • Kye-Il Joo
  • Leslie Ziegler
  • Pin WangEmail author
Research Paper



The purpose of this study was to investigate the potential of a T-cell-related targeting method using a lentiviral vector-based gene delivery system.

Materials and Methods

A lentiviral vector system was constructed by co-incorporating an anti-CD3 antibody (OKT3) and a fusogen into individual viral particles. The incorporation of OKT3 and fusogen was analyzed using confocal microscopy and the in vitro transduction efficiency was evaluated using flow cytometry. Blocking reagents (ammonium chloride (NH4Cl) and soluble OKT3 antibody) were added into vector supernatants during transduction to study the mechanism of this two-molecule targeting strategy. To demonstrate the ability of targeted transduction in vivo, Jurkat.CD3 cells were xenografted subcutaneously into the right flank of each mouse and the lentiviral vector was injected subcutaneously on both sides of each mouse 8 h post-injection. Subsequently, the reporter gene (firefly luciferase) expression was monitored using a noninvasive bioluminescence imaging system.


By co-displaying OKT3 and fusogen on the single lentiviral surface, we could achieve targeted delivery of genes to CD3-positive T-cells both in vitro and in vivo.


These results suggest the potential utility of this engineered lentiviral system as a new tool for cell type-directed gene delivery.


CD3 antigen gene therapy lentiviral vectors targeted gene delivery 



We thank April Tai, Lili Yang and Steven Froelich for critical reading of the manuscript, and the USC Norris Center Cell and Tissue Imaging Core. This work was supported by a National Institute of Health grant. The following reagents was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Monoclonal Antibody to HIV-1 p24 (AG3.0) from Dr. Jonathan Allan.


  1. 1.
    Y. Chernajovsky, G. Adams, K. Triantaphyllopoulos, M. F. Ledda, and O. L. Podhajcer. Pathogenic lymphoid cells engineered to express TGF beta 1 ameliorate disease in a collagen-induced arthritis model. Gene. Ther. 4:553–559 (1997) doi: 10.1038/ Scholar
  2. 2.
    A. Aiuti, S. Vai, A. Mortellaro, G. Casorati, F. Ficara, G. Andolfi, G. Ferrari, A. Tabucchi, F. Carlucci, H. D. Ochs, L. D. Notarangelo, M. G. Roncarolo, and C. Bordignon. Immune reconstitution in ADA-SCID after PBL gene therapy and discontinuation of enzyme replacement. Nat. Med. 8:423–425 (2002) doi: 10.1038/nm0502-423.PubMedCrossRefGoogle Scholar
  3. 3.
    E. Verhoeyen, V. Dardalhon, O. Ducrey-Rundquist, D. Trono, N. Taylor, and F. L. Cosset. IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes. Blood. 101:2167–2174 (2003) doi: 10.1182/blood-2002-07-2224.PubMedCrossRefGoogle Scholar
  4. 4.
    W. R. Drobyski, H. C. Morse 3rd, W. H. Burns, J. T. Casper, and G. Sandford. Protection from lethal murine graft-versus-host disease without compromise of alloengraftment using transgenic donor T cells expressing a thymidine kinase suicide gene. Blood. 97:2506–2513 (2001) doi: 10.1182/blood.V97.8.2506.PubMedCrossRefGoogle Scholar
  5. 5.
    M. Maurice, E. Verhoeyen, P. Salmon, D. Trono, S. J. Russell, and F. L. Cosset. Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell-activating polypeptide. Blood. 99:2342–2350 (2002) doi: 10.1182/blood.V99.7.2342.PubMedCrossRefGoogle Scholar
  6. 6.
    R. A. Morgan, M. E. Dudley, J. R. Wunderlich, M. S. Hughes, J. C. Yang, R. M. Sherry, R. E. Royal, S. L. Topalian, U. S. Kammula, N. P. Restifo, Z. Zheng, A. Nahvi, C. R. de Vries, L. J. Rogers-Freezer, S. A. Mavroukakis, and S. A. Rosenberg. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science. 314:126–129 (2006) doi: 10.1126/science.1129003.PubMedCrossRefGoogle Scholar
  7. 7.
    B. L. Levine, L. M. Humeau, J. Boyer, R. R. MacGregor, T. Rebello, X. Lu, G. K. Binder, V. Slepushkin, F. Lemiale, J. R. Mascola, F. D. Bushman, B. Dropulic, and C. H. June. Gene transfer in humans using a conditionally replicating lentiviral vector. Proc. Natl. Acad. Sci. U.S.A. 103:17372–17377 (2006) doi: 10.1073/pnas.0608138103.PubMedCrossRefGoogle Scholar
  8. 8.
    M. Sadelain, I. Riviere, and R. Brentjens. Targeting tumours with genetically enhanced T lymphocytes. Nat. Rev. Cancer. 3:35–45 (2003) doi: 10.1038/nrc971.PubMedCrossRefGoogle Scholar
  9. 9.
    M. T. Stephan, V. Ponomarev, R. J. Brentjens, A. H. Chang, K. V. Dobrenkov, G. Heller, and M. Sadelain. T cell-encoded CD80 and 4–1BBL induce auto- and transcostimulation, resulting in potent tumor rejection. Nat. Med. 13:1440–1449 (2007) doi: 10.1038/nm1676.PubMedCrossRefGoogle Scholar
  10. 10.
    T. N. Schumacher. T-cell-receptor gene therapy. Nat. Rev. Immunol. 2:512–519 (2002) doi: 10.1038/nri841.PubMedCrossRefGoogle Scholar
  11. 11.
    O. J. Muller, F. Kaul, M. D. Weitzman, R. Pasqualini, W. Arap, J. A. Kleinschmidt, and M. Trepel. Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors. Nat. Biotechnol. 21:1040–1046 (2003) doi: 10.1038/nbt856.PubMedCrossRefGoogle Scholar
  12. 12.
    D. G. Miller, M. A. Adam, and A. D. Miller. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol. Cell Biol. 10:4239–4242 (1990).PubMedGoogle Scholar
  13. 13.
    L. Naldini, U. Blomer, P. Gallay, D. Ory, R. Mulligan, F. H. Gage, I. M. Verma, and D. Trono. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 272:263–267 (1996) doi: 10.1126/science.272.5259.263.PubMedCrossRefGoogle Scholar
  14. 14.
    E. Costello, M. Munoz, E. Buetti, P. R. Meylan, H. Diggelmann, and M. Thali. Gene transfer into stimulated and unstimulated T lymphocytes by HIV-1-derived lentiviral vectors. Gene Ther. 7:596–604 (2000) doi: 10.1038/ Scholar
  15. 15.
    D. B. Kohn. Lentiviral vectors ready for prime-time. Nat. Biotechnol. 25:65–66 (2007) doi: 10.1038/nbt0107-65.PubMedCrossRefGoogle Scholar
  16. 16.
    L. E. Ailles, and L. Naldini. HIV-1-derived lentiviral vectors. Curr. Top. Microbiol. Immunol. 261:31–52 (2002).PubMedGoogle Scholar
  17. 17.
    J. Cronin, X. Y. Zhang, and J. Reiser. Altering the tropism of lentiviral vectors through pseudotyping. Curr. Gene Ther. 5:387–398 (2005) doi: 10.2174/1566523054546224.PubMedCrossRefGoogle Scholar
  18. 18.
    V. Sandrin, S. J. Russell, and F. L. Cosset. Targeting retroviral and lentiviral vectors. Curr. Top. Microbiol. Immunol. 281:137–178 (2003).PubMedGoogle Scholar
  19. 19.
    E. Verhoeyen, and F. L. Cosset. Surface-engineering of lentiviral vectors. J. Gene Med. 6(Suppl 1):S83–S94 (2004) doi: 10.1002/jgm.494.PubMedCrossRefGoogle Scholar
  20. 20.
    R. Waehler, S. J. Russell, and D. T. Curiel. Engineering targeted viral vectors for gene therapy. Nat. Rev. Genet. 8:573–587 (2007) doi: 10.1038/nrg2141.PubMedCrossRefGoogle Scholar
  21. 21.
    D. Lavillette, S. J. Russell, and F. L. Cosset. Retargeting gene delivery using surface-engineered retroviral vector particles. Curr. Opin. Biotechnol. 12:461–466 (2001) doi: 10.1016/S0958-1669(00)00246-9.PubMedCrossRefGoogle Scholar
  22. 22.
    S. Ager, B. H. Nilson, F. J. Morling, K. W. Peng, F. L. Cosset, and S. J. Russell. Retroviral display of antibody fragments; interdomain spacing strongly influences vector infectivity. Hum. Gene Ther. 7:2157–2164 (1996) doi: 10.1089/hum.1996.7.17-2157.PubMedCrossRefGoogle Scholar
  23. 23.
    M. Marin, D. Noel, S. Valsesia-Wittman, F. Brockly, M. Etienne-Julan, S. Russell, F. L. Cosset, and M. Piechaczyk. Targeted infection of human cells via major histocompatibility complex class I molecules by Moloney murine leukemia virus-derived viruses displaying single-chain antibody fragment-envelope fusion proteins. J. Virol. 70:2957–2962 (1996).PubMedGoogle Scholar
  24. 24.
    S. Chowdhury, K. A. Chester, J. Bridgewater, M. K. Collins, and F. Martin. Efficient retroviral vector targeting of carcinoembryonic antigen-positive tumors. Mol. Ther. 9:85–92 (2004) doi: 10.1016/j.ymthe.2003.10.004.PubMedCrossRefGoogle Scholar
  25. 25.
    S. Funke, A. Maisner, M. D. Muhlebach, U. Koehl, M. Grez, R. Cattaneo, K. Cichutek, and C. J. Buchholz. Targeted cell entry of lentiviral vectors. Mol. Ther. 16:1427–1436 (2008) doi: 10.1038/mt.2008.128.PubMedCrossRefGoogle Scholar
  26. 26.
    K. Morizono, G. Bristol, Y. M. Xie, S. K. Kung, and I. S. Chen. Antibody-directed targeting of retroviral vectors via cell surface antigens. J. Virol. 75:8016–8020 (2001) doi: 10.1128/JVI.75.17.8016-8020.2001.PubMedCrossRefGoogle Scholar
  27. 27.
    K. Morizono, Y. Xie, G. E. Ringpis, M. Johnson, H. Nassanian, B. Lee, L. Wu, and I. S. Chen. Lentiviral vector retargeting to P-glycoprotein on metastatic melanoma through intravenous injection. Nat. Med. 11:346–352 (2005) doi: 10.1038/nm1192.PubMedCrossRefGoogle Scholar
  28. 28.
    P. Roux, P. Jeanteur, and M. Piechaczyk. A versatile and potentially general approach to the targeting of specific cell types by retroviruses: application to the infection of human cells by means of major histocompatibility complex class I and class II antigens by mouse ecotropic murine leukemia virus-derived viruses. Proc. Natl. Acad. Sci. U.S.A. 86:9079–9083 (1989) doi: 10.1073/pnas.86.23.9079.PubMedCrossRefGoogle Scholar
  29. 29.
    A. L. Boerger, S. Snitkovsky, and J. A. Young. Retroviral vectors preloaded with a viral receptor-ligand bridge protein are targeted to specific cell types. Proc. Natl. Acad. Sci. U.S.A. 96:9867–9872 (1999) doi: 10.1073/pnas.96.17.9867.PubMedCrossRefGoogle Scholar
  30. 30.
    L. Yang, L. Bailey, D. Baltimore, and P. Wang. Targeting lentiviral vectors to specific cell types in vivo. Proc. Natl. Acad. Sci. U.S.A. 103:11479–11484 (2006) doi: 10.1073/pnas.0604993103.PubMedCrossRefGoogle Scholar
  31. 31.
    A. H. Lin, N. Kasahara, W. Wu, R. Stripecke, C. L. Empig, W. F. Anderson, and P. M. Cannon. Receptor-specific targeting mediated by the coexpression of a targeted murine leukemia virus envelope protein and a binding-defective influenza hemagglutinin protein. Hum. Gene Ther. 12:323–332 (2001) doi: 10.1089/10430340150503957.PubMedCrossRefGoogle Scholar
  32. 32.
    H. Yang, L. Zeigler, K. I. Joo, T. Cho, Y. Lei, and P. Wang. Gamma-retroviral vectors enveloped with an antibody and an engineered fusogenic protein achieved antigen-specific targeting. Biotechnol. Bioeng. 19:861–872 (2008).Google Scholar
  33. 33.
    C. Lois, E. J. Hong, S. Pease, E. J. Brown, and D. Baltimore. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science. 295:868–872 (2002) doi: 10.1126/science.1067081.PubMedCrossRefGoogle Scholar
  34. 34.
    A. L. Szymczak, C. J. Workman, Y. Wang, K. M. Vignali, S. Dilioglou, E. F. Vanin, and D. A. Vignali. Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat. Biotechnol. 22:589–594 (2004) doi: 10.1038/nbt957.PubMedCrossRefGoogle Scholar
  35. 35.
    J. Fang, J.-J. Qian, S. Yi, T. C. Harding, G. H. Tu, M. VanRoey, and K. Jooss. Stable antibody expression at therapeutic levels using the 2A peptide. Nat. Biotech. 23:584–590 (2005) doi: 10.1038/nbt1087.CrossRefGoogle Scholar
  36. 36.
    K. I. Joo, and P. Wang. Visualization of targeted transduction by engineered lentiviral vectors. Gene Ther. 15:1348–1396 (2008) doi: 10.1038/gt.2008.87.CrossRefGoogle Scholar
  37. 37.
    A. B. Cosimi, R. C. Burton, R. B. Colvin, G. Goldstein, F. L. Delmonico, M. P. LaQuaglia, N. Tolkoff-Rubin, R. H. Rubin, J. T. Herrin, and P. S. Russell. Treatment of acute renal allograft rejection with OKT3 monoclonal antibody. Transplantation. 32:535–539 (1981) doi: 10.1097/00007890-198112000-00018.PubMedCrossRefGoogle Scholar
  38. 38.
    A. B. Cosimi, R. B. Colvin, R. C. Burton, R. H. Rubin, G. Goldstein, P. C. Kung, W. P. Hansen, F. L. Delmonico, and P. S. Russell. Use of monoclonal antibodies to T-cell subsets for immunologic monitoring and treatment in recipients of renal allografts. N. Engl. J. Med. 305:308–314 (1981).PubMedGoogle Scholar
  39. 39.
    M. G. Rudolph, R. L. Stanfield, and I. A. Wilson. How TCRs bind MHCs, peptides, and coreceptors. Annu. Rev. Immunol. 24:419–466 (2006) doi: 10.1146/annurev.immunol.23.021704.115658.PubMedCrossRefGoogle Scholar
  40. 40.
    M. Reth. Antigen receptors on B lymphocytes. Annu. Rev. Immunol. 10:97–121 (1992) doi: 10.1146/annurev.iy.10.040192.000525.PubMedCrossRefGoogle Scholar
  41. 41.
    Y. E. Lu, T. Cassese, and M. Kielian. The cholesterol requirement for sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence. J. Virol. 73:4272–4278 (1999).PubMedGoogle Scholar
  42. 42.
    I. Mellman, R. Fuchs, and A. Helenius. Acidification of the endocytic and exocytic pathways. Annu. Rev. Biochem. 55:663–700 (1986) doi: 10.1146/ Scholar
  43. 43.
    M. Kielian, and F. A. Rey. Virus membrane-fusion proteins: more than one way to make a hairpin. Nat. Rev. Microbiol. 4:67–76 (2006) doi: 10.1038/nrmicro1326.PubMedCrossRefGoogle Scholar
  44. 44.
    D. L. Gibbons, M. C. Vaney, A. Roussel, A. Vigouroux, B. Reilly, J. Lepault, M. Kielian, and F. A. Rey. Conformational change and protein–protein interactions of the fusion protein of Semliki Forest virus. Nature. 427:320–325 (2004) doi: 10.1038/nature02239.PubMedCrossRefGoogle Scholar
  45. 45.
    M. Umashankar, C. Sanchez-San Martin, M. Liao, B. Reilly, A. Guo, G. Taylor, and M. Kielian. Differential cholesterol binding by class II fusion proteins determines membrane fusion properties. J. Virol. 82:9245–9253 (2008) doi: 10.1128/JVI.00975-08.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Haiguang Yang
    • 1
  • Kye-Il Joo
    • 1
  • Leslie Ziegler
    • 1
  • Pin Wang
    • 1
    Email author
  1. 1.Mork Family Department of Chemical Engineering and Materials ScienceUniversity of Southern CaliforniaLos AngelesUSA

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