Somatic Cell and Molecular Genetics

, Volume 26, Issue 1–6, pp 147–158

Safety Considerations in Vector Development

  • John C. Kappes
  • Xiaoyun Wu


The inadvertent production of replication competent retrovirus (RCR) constitutes the principal safety concern for the use of lentiviral vectors in human clinical protocols. Because of limitations in animal models to evaluate lentiviral vectors for their potential to recombine and induce disease, the vector design itself should ensure against the emergence of RCR in vivo. Issues related to RCR generation and one approach to dealing with this problem are discussed in this chapter. To assess the risk of generating RCR, a highly sensitive biological assay was developed to specifically detect vector recombination in transduced cells. Analysis of lentiviral vector stocks has shown that recombination occurs during reverse transcription in primary target cells. Rejoining of viral protein-coding sequences of the packaging construct and cis-acting sequences of the vector was demonstrated to generate env-minus recombinants (LTR-gag-pol-LTR). Mobilization of recombinant lentiviral genomes was also demonstrated but was dependent on pseudotyping of the vector core with an exogenous envelope protein. 5′ sequence analysis has demonstrated that recombinants consist of U3, R, U5, and the ψ packaging signal joined with an open gag coding region. Analysis of the 3′ end has mapped the point of vector recombination to the poly(A) tract of the packaging construct's mRNA. The state-of-the-art third generation packaging construct and SIN vector also have been shown to generate env-minus proviral recombinants capable of mobilizing retroviral DNA when pseudotyped with an exogenous envelope protein. A new class of HIV-based vector (trans-vector) was recently developed that splits the gag-pol component of the packaging construct into two parts: one that expresses Gag/Gag-Pro and another that expresses Pol (RT and IN) fused with Vpr. Unlike other lentiviral vectors, the trans-vector has not been shown to form recombinants capable of DNA mobilization. These results indicate the trans-vector design prevents the generation of env-minus recombinant lentivirus containing a functional gag-pol structure (LTR-gag-pol-LTR), which is absolutely required for retroviral DNA mobilization and the emergence of RCR. Quality assurance based on monitoring for RCR may have limitations as a predictor of safety in vivo, especially in the long term. The demonstration of lentivirus infection via alternative entry mechanisms supports this notion. Therefore, the approach of monitoring trans-vector stocks for env-minus recombinant virus in vitro as a surrogate marker for the possible emergence of RCR in vivo should represent a significant advancement in vector safety quality assurance.


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  1. 1.
    Miller A. Retroviral vectors. Cur Top Microbiol 1992; 158:1-24.Google Scholar
  2. 2.
    Vile RG, Russell SJ. Retroviruses as vectors. Br Med Bull 1995; 51:12-30.Google Scholar
  3. 3.
    Lewis P, Emerman M. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J Virol 1994; 68:510-516.Google Scholar
  4. 4.
    Roe T, Reynolds T, Yu G et al. Integration of murine leukemia virus DNA depends on mitosis. EMBO J 1993; 12:2099-2108.Google Scholar
  5. 5.
    Mann R, Mulligan RC, Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 1983; 33:153-159.Google Scholar
  6. 6.
    Miller MD, Farnet CM, Bushman FD. Human immunodeficiency virus type 1 preintegration complexes: Studies of organization and composition. J Virol 1997; 71:5382-5390.Google Scholar
  7. 7.
    Anderson WF, McGarrity GJ, Moen RC. Report to the NIH recombinant advisory committee on murine replication-competent retrovirus (RCR) assays. Hum Gene ther 1993;4:311-321.Google Scholar
  8. 8.
    Wilson CA, Ng TH, Miller AE. Evaluation of recommendations for replication-competent retrovirus testing associated with use of retroviral vectors. Hum Gene ther 1997; 8:869-874.Google Scholar
  9. 9.
    Miller AD. Development and application of retroviral vectors. In: Coffin JM, Hughes SH, Varmus HE, eds. Retroviruses. Cold Spring Harbor: Cold Spring Harbor Laboratory, 1997:437-474.Google Scholar
  10. 10.
    Donahue RE, Kessler SW, Bodine D et al. Helper virus induced t cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J Exp Med 1992; 176:1125-1135.Google Scholar
  11. 11.
    Otto E, Jones-Trower A, Vanin EF et al. Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences. Hum Gene Ther 1994; 5:567-575.Google Scholar
  12. 12.
    Vanin EF, Kaloss M, Broscius C et al. Characterization of replication-competent retroviruses from nonhuman primates with virus-induced t-cell lymphomas and observations regarding the mechanism of oncogenesis. J Virol 1994; 68:4241-4250.Google Scholar
  13. 13.
    Purcell DFJ, Broscius CM, Vanin E et al. An array of murine leukemia virus-related elements is transmitted and expressed in a primate recipient of retroviral gene transfer. J Virol. 1996; 70:887-897.Google Scholar
  14. 14.
    Bestwick RK, Kozak SL, Kabat D. Overcoming interference to retroviral superinfection results in amplified expression and transmission of cloned genes. Proc Natl Acad Sci USA 1988; 85:5404-5408.Google Scholar
  15. 15.
    Bodine DM, McDonagh KT, Brandt SJ et al. Development of a high-titer retrovirus product line capable of gene transfer into rhesus monkey hematopoietic stem cells. Proc Natl Acad Sci USA1990; 87:3738-3742.Google Scholar
  16. 16.
    Chong H, Vile RG. Replication-competent retrovirus produced by a “split-function” third generation amphotropic packaging cell line. Gene Ther 1996;3:624-629.Google Scholar
  17. 17.
    Markowitz D, Goff S, Bank A. Construction and use of a safe and efficient amphotropic packaging cell line. Virology 1988; 167:400-406.Google Scholar
  18. 18.
    Chong H, Starkey W, Vile RG. A replication-competent retrovirus arising from a split-function packaging cell line was renerated by recombination events between the vector, one of the packaging constructs, and endogenous retroviral sequences. J Virol. 1998; 72:2663-2670.Google Scholar
  19. 19.
    Rigg RJ, Chen J, Dando JS et al. A novel human amphotropic packaging cell line: High titer, complement resistance, and improved safety. Virology 1996; 218:290-295.Google Scholar
  20. 20.
    Boeke JD, Stoye JP. Retrotransposons, endogenous retroviruses, and the evolution of retroelements. In: Coffin JM, Hughes SH, Varmus HE, eds. Retroviruses. Plainview: Cold Spring Harbor Laboratory Press, 1997:343-435.Google Scholar
  21. 21.
    Bukrinsky MI, Sharova N, Dempsey MP et al. Active nuclear import of human immunodeficiency virus type 1 preintegration complexes. Proc Natl Acad Sci USA 1992; 89:6580-6584.Google Scholar
  22. 22.
    Lewis PF, Hansel M, Emerman M. Human immunodeficiency virus infection of cells arrested in the cell cycle. EMBO J 1992; 11:3053-3058.Google Scholar
  23. 23.
    Amado RG, Chen ISY. Lentiviral vectors-the promise of gene therapy within reach? Science 1999;285:674-676.Google Scholar
  24. 24.
    Kordower JH, Emborg ME, Bloch J et al. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science 2000; 290:767-773.Google Scholar
  25. 25.
    Miyoshi H, Smith KA, Mosier DE et al. Transduction of human CD34+ cells that mediate longterm engraftment of NOD/SCID mice by HIV vectors. Science 1999; 283:682-686.Google Scholar
  26. 26.
    Trono D. Lentiviral vectors: Turning a deadly foe into a therapeutic agent. Gene Ther 2000;7:20-23.Google Scholar
  27. 27.
    May C, Rivella S, Callegari J et al. Therapeutic heamoglobin synthesis in ?-thalassaemic mice expressing lentivirus-encoded human ?-globin. Nature 2000; 406:82-86.Google Scholar
  28. 28.
    Kafri T, VAN Praag H, Ouyang L et al. A packaging cell line for lentivirus vectors. J Virol 1999;73:576-584.Google Scholar
  29. 29.
    Naldini L, Blomer U, Gallay P et al. In vivo gene delivery and stable gene transduction of nondividing cells by a lentivirus vector. Science 1996; 272:263-267.Google Scholar
  30. 30.
    Dull T, Zufferey R, Kelly M et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998; 72:8463-8471.Google Scholar
  31. 31.
    Kim VN, Mitrophanous K, Kingsman SM et al. Minimal Requirement for a lentivirus vector based on human immunodeficiency virus type 1. J Virol 1998; 72:811-816.Google Scholar
  32. 32.
    Zufferey R, Nagy D, Mandel RJ et al. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnology 1997; 15:871-875.Google Scholar
  33. 33.
    Gasmi M, Glynn J, Jin M-J et al. Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors. J Virol 1999; 73(3):1828-1834.Google Scholar
  34. 34.
    Miyoshi H, Blomer U, Takahashi M et al. Development of a self-inactivating lentivirus vector. J Virol 1998; 72:8150-8157.Google Scholar
  35. 35.
    Zufferey R, Dull T, Mandel RJ et al. Self-inactivating lentivirus vector for safe and efficient in vivo Gene Therapy. J Virol 1998; 72:9873-9880.Google Scholar
  36. 36.
    Wu X, Wakefield KJ, Liu H et al. Development of a novel trans-lentiviral vector that affords predictable safety. Mol Ther 2000; 2:47-55.Google Scholar
  37. 37.
    Huang CC, Hay N, Bishop JM. The role of RNA molecules in transduction of the proto-oncogene c-fps. Cell 1986; 44:935-940.Google Scholar
  38. 38.
    Raines MA, Maihle NJ, Mascovici C et al. Mechanism of c-erbB transduction: Newly released transducing viruses retain poly(A) tracts of erbB transcripts and encode C-terminally intact erbB proteins. J Virol 1988; 62:2437-2443.Google Scholar
  39. 39.
    Liu H, Wu X, Xiao H et al. Incorporation of functional human immunodeficiency virus type 1 integrase into virions independent of the Gag-Pol precursor protein. J Virol 1997; 71:7701-7710.Google Scholar
  40. 40.
    Wu X, Liu H, Xiao H et al. Human immunodeficiency virus type 1 integrase protein promotes reverse transcription through specific interactions with the nucleoprotein reverse transcription complex. J Virol 1999; 73:2126-2135.Google Scholar
  41. 41.
    Wu X, Liu H, Xiao H et al. Functional RT and IN incorporated into HIV-1 particles independently of the Gag-Pol precursor protein. EMBO J 1997; 16:5113-5122.Google Scholar
  42. 42.
    Wu X, Liu Ho, Xiao H et al. Targeting foreign proteins to human immunodeficiency virus particles via fusion with Vpr and Vpx. J Virol 1995; 69:3389-3398.Google Scholar
  43. 43.
    Chen WY, Wu X, Levasseur DN et al. Lentiviral vector transduction of hematopoietic stem cells that mediate long-term reconstitution of lethally irradiate mice. Stem Cells 2000; 18:352-359.Google Scholar
  44. 44.
    Abonour R, Williams DA, Einhorn L et al. Efficient retrovius-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells. Nat Med 2000; 6(6):652-628.Google Scholar
  45. 45.
    Miyoshi H, Takahashi M, Gage FH et al. Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc Natl Acad Sci USA 1997; 94:10319-10323.Google Scholar
  46. 46.
    Takahashi M, Miyoshi H, Verma IM et al. Rescue from photoreceptor degeneration in the rd mouse by human immunodeficiency virus vector-mediated gene transfer. J Virol 1999; 73:7812.Google Scholar
  47. 47.
    Endres MJ, Jaffer S, Haggarty B et al. Targeting of HIV-and SIV-infected cells by CD4-chemokine receptor pseudotypes. Science 1997; 278:1462-1464.Google Scholar
  48. 48.
    Mebatsion T, Finke S, Weiland F et al. A CXCR4/CD4 pseudotype rhabdovirus that selectively infects HIV-1 envelope protein-expressing cells. Cell 1997; 90:841-847.Google Scholar
  49. 49.
    Schnell MJ, Johnson JE, Buonocore L et al. Construction of a novel virus that targets HIV-1-infected cells and controls HIV-1 infection. Cell 1997; 90:849-857.Google Scholar
  50. 50.
    Blobel CP, Wolfsberg TG, Turck CW et al. A potential fusion peptide and an integrin ligand domain in a protein active in sperm-egg fusion. Nat Med 1992; 356:248-252.Google Scholar
  51. 51.
    Pang S, Yu D, An D-S et al. Human immunodeficiency virus env-independent infection of human CD4-cells. J Virol 2000; 74:10994-11000.Google Scholar
  52. 52.
    Padow M, Lai L, Fisher RJ et al. Analysis of human immunodeficiency virus type 1 containing HERV-K protease. AIDS Res Hum Retroviruses 2000; 16(18):1973-1980.Google Scholar
  53. 53.
    Olsen JC. Gene transfer vectors derived from equine infectious anemia virus. Gene Ther 1998;5:1481-1487.Google Scholar
  54. 54.
    Poeschla EF, Wong-Staal F, Looney DJ. Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors. Nat Med 1998; 4:354-357.Google Scholar

Copyright information

© Plenum Publishing Corporation 2001

Authors and Affiliations

  • John C. Kappes
    • 1
  • Xiaoyun Wu
    • 1
  1. 1.Department of MedicineUniversity of Alabama at BirminghamBirmingham

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