Gene therapy: trials and tribulations

The art and science of gene therapy has received much attention of late. The tragic death of 18-year-old Jesse Gelsinger, a volunteer in a Phase I clinical trial, has overshadowed the successful treatment of three children suffering from a rare but fatal immunological disease. In the light of the success and tragedy, it is timely to consider the challenges faced by gene therapy — a novel form of molecular medicine that may be poised to have an important impact on human health in the new millennium.

Key Points

  • A key problem in gene therapy is the lack of a vector system that fulfils all the requirements for safety and efficacy.

  • Viral vectors are the most promising vectors at this time. Integrating viruses are based on retrovirus, lentivirus and adeno-associated virus. Some vectors are based on adenovirus — a non-integrating virus.

  • Immunological barriers are a problem for all vectors, but particularly for adenoviral vectors.

  • The death of Jesse Gelsinger in a gene therapy Phase I clinical trial has overshadowed some recent successes in gene therapy in animal models and notably in humans with a form of severe combined immune deficiency.

  • The next phase of gene therapy will be focused on targeted and regulated expression of the therapeutic gene.

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Figure 1: Retrovirus-based vectors.
Figure 2: In vivo gene delivery into mice and rats using lentiviral vectors.
Figure 3: Adeno-associated viral vectors.
Figure 4: Regulation of gene expression.
Figure 5: Regulated expression of a gene therapy vector.

References

  1. 1

    Li, S. & Huang, L. Nonviral gene therapy: promises and challenges. Gene Ther. 7, 31– 34 (2000).

  2. 2

    Templeton, N. S. & Lasic, D. D. (eds) Gene Therapy: Therapeutic Mechanisms and Strategies (Marcel Dekker, Inc., New York, 2000).This book has several excellent chapters on viral and non-viral vectors written by experts in the field. It also describes a number of therapeutic approaches to diseases and requirements for regulatory affairs.

  3. 3

    Friedmann, T. (ed.) The Development of Human Gene Therapy (Cold Spring Harbor Laboratory Press, New York, 1999).

  4. 4

    Verma, I. M. Gene therapy. Sci. Am. 263, 68– 72 (1990).

  5. 5

    Anderson, W. F. Human gene therapy. Nature 392, 25– 30 (1998).

  6. 6

    Mulligan, R. C. The basic science of gene therapy. Science 260, 926–932 (1993).

  7. 7

    Miller, A. D. Human gene therapy comes of age. Nature 357, 455–460 (1992).

  8. 8

    Markowitz, D., Goff, S. & Bank, A. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J. Virol. 62, 1120– 1124 (1988).

  9. 9

    Danos, O. & Mulligan, R. C. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl Acad. Sci. USA 85, 6460– 6464 (1988).

  10. 10

    Burns, J. C., Friedmann, T., Driever, W., Burrascano, M. & Yee, J. K. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and non-mammalian cells. Proc. Natl Acad. Sci. USA 90, 8033–8037 (1993).Use of vesicular stomatitis virus glycoprotein (VSVG) to replace the envelope protein in MLV vectors. The resulting chimeric virus, containing VSVG protein, is pantropic and can be concentrated by ultracentrifugation.

  11. 11

    Yu, S. F. et al. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc. Natl Acad. Sci. USA 83, 3194–3198 (1986).

  12. 12

    Morgan, R. A. & Anderson, W. F. Human gene therapy. Annu. Rev. Biochem. 62, 191–217 (1993).

  13. 13

    St Louis, D. & Verma, I. M. An alternative approach to somatic cell gene therapy. Proc. Natl Acad. Sci. USA 85, 3150–3154 (1988). On transplantation of retrovirally-transduced mouse fibroblasts, producing and secreting human factor IX protein, the transcription of the transgene is `shut off'.

  14. 14

    Palmer, T. D., Rosman, G. J., Osborne, W. R. & Miller, A. D. Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes. Proc. Natl Acad. Sci. USA 88, 1330–1334 ( 1991).

  15. 15

    Lewis, P., Hensel, M. & Emerman, M. Human immunodeficiency virus infection of cells arrested in the cell cycle. EMBO J. 11, 3053– 3058 (1992).

  16. 16

    Bukrinsky, M. I. et al. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature 365, 666–669 (1993).

  17. 17

    Frankel, A. D. & Young, J. A. HIV-1: fifteen proteins and an RNA. Annu. Rev. Biochem. 67, 1–25 (1998).

  18. 18

    Naldini, L. et al. In vivo gene delivery and stable transduction of non-dividing cells by a lentiviral vector. Science 272, 263–267 (1996).The first use of a lentiviral vector to transduce genes in vitro and in vivo in non-dividing cells. The lentiviral vector was packaged in VSVG envelope protein to expand the host range.

  19. 19

    Kafri, T., Blomer, U., Peterson, D. A., Gage, F. H. & Verma, I. M. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nature Genet. 17, 314–317 ( 1997).

  20. 20

    Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L. & Trono, D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nature Biotechnol. 15 , 871–875 (1997).

  21. 21

    Zennou, V. et al. HIV-1 genome nuclear import is mediated by a central DNA flap . Cell 101, 173–185 (2000).

  22. 22

    Follenzi, A., Ailles, L. E., Bakovic, S., Geuna, M. & Naldini, L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences . Nature Genet. 25, 217– 222 (2000).

  23. 23

    Zufferey, R., Donello, J. E., Trono, D. & Hope, T. J. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J. Virol. 73, 2886–2892 ( 1999).

  24. 24

    Miyoshi, H., Blomer, U., Takahashi, M., Gage, F. H. & Verma, I. M. Development of a self-inactivating lentivirus vector. J. Virol. 72, 8150– 8157 (1998).

  25. 25

    Zufferey, R. et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72, 9873–9880 (1998).

  26. 26

    Johnston, J. C. et al. Minimum requirements for efficient transduction of dividing and nondividing cells by feline immunodeficiency virus vectors. J. Virol. 73, 4991–5000 (1999).

  27. 27

    Schnell, T., Foley, P., Wirth, M., Munch, J. & Uberla, K. Development of a self-inactivating, minimal lentivirus vector based on simian immunodeficiency virus. Hum. Gene Ther. 11, 439–447 ( 2000).

  28. 28

    Poeschla, E. M., Wong-Staal, F. & Looney, D. J. Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors. Nature Med. 4, 354–357 ( 1998).

  29. 29

    Mitrophanous, K. et al. Stable gene transfer to the nervous system using a non-primate lentiviral vector. Gene Ther. 6, 1808– 1818 (1999).

  30. 30

    Carter, P. J. & Samulski, R. J. Adeno-associated viral vectors as gene delivery vehicles. Int. J. Mol. Med. 6, 17–27 (2000).

  31. 31

    Berns, K. I. in Fields Virology (eds Fields, B. N., Knipe, D. M. & Howley P. M.,) 2173–2198 (Lippincott–Raven, Philadelphia, 1996).

  32. 32

    Kotin, R. M. et al. Site-specific integration by adeno-associated virus. Proc. Natl Acad. Sci. USA 87, 2211– 2215 (1990).

  33. 33

    Inoue, N., Hirata, R. K. & Russell, D. W. High-fidelity correction of mutations at multiple chromosomal positions by adeno-associated virus vectors. J. Virol. 73, 7376–7380 ( 1999).

  34. 34

    Summerford, C. & Samulski, R. J. Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J. Virol. 72, 1438– 1445 (1998).

  35. 35

    Girod, A. et al. Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2. Nature Med. 5, 1052–1056 (1999).

  36. 36

    Malik, P. et al. Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line. J. Virol. 71, 1776– 1783 (1997).

  37. 37

    Kay, M. A. et al. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nature Genet. 24, 257–261 ( 2000).Gives the first hints of successful gene therapy in haemophilia B patients by using recombinant adeno-associated viral vectors. Expression of transduced factor IX could be detected in one patient.

  38. 38

    Yan, Z., Zhang, Y., Duan, D. & Engelhardt, J. F. Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy. Proc. Natl Acad. Sci. USA 97, 6716– 6721 (2000).

  39. 39

    Nakai, H., Storm, T. A. & Kay, M. A. Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors. Nature Biotechnol. 18, 527–532 (2000).

  40. 40

    Shenk, T. in Fields Virology (eds Fields, B. N., Knipe D. M. & Howley, P. M.) 2111–2148 (Lippincott–Raven, Philadelphia, 1996).

  41. 41

    Yeh, P. & Perricaudet, M. Advances in adenoviral vectors: from genetic engineering to their biology. FASEB J. 11, 615–623 (1997).

  42. 42

    Kochanek, S. et al. A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and β-galactosidase. Proc. Natl Acad. Sci. USA 93, 5731–5736 (1996).

  43. 43

    Parks, R. J. et al. A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc. Natl Acad. Sci. USA 93, 13565– 13570 (1996).References 42 and 43 describe the generation of new adenoviral, `gutless', vectors in which all the viral genes required for viral propagation are provided in trans . The gutless vectors show long-term expression of the transgene.

  44. 44

    Morral, N. et al. Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons. Proc. Natl Acad. Sci. USA 96, 12816–12821 (1999).

  45. 45

    Dai, Y. et al. Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression. Proc. Natl Acad. Sci. USA 92, 1401–1405 (1995).

  46. 46

    Kafri, T. et al. Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy. Proc. Natl Acad. Sci. USA 95, 11377 –11382 (1998).Even physically inactivated adenoviral particles can generate a cytotoxic T-cell response, raising concerns about adenoviral vectors as suitable tools for long-term gene therapy.

  47. 47

    Tripathy, S. K., Black, H. B., Goldwasser, E. & Leiden, J. M. Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors. Nature Med. 2, 545–550 ( 1996).

  48. 48

    Wagner, R. R., & Rose, J. K. in Fields Virology (eds Fields, B. N., Knipe D. M. & Howley, P. M.) 1121– 1136 (Lippincott–Raven, Philadelphia, 1996).

  49. 49

    Chirmule, N. et al. Humoral immunity to adeno-associated virus type 2 vectors following administration to murine and nonhuman primate muscle. J. Virol. 74, 2420–2425 (2000).

  50. 50

    Halbert, C. L., Rutledge, E. A., Allen, J. M., Russell, D. W. & Miller, A. D. Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes. J. Virol. 74, 1524–1532 (2000).

  51. 51

    Fields, P. A. et al. Role of vector in activation of T cell subsets in immune responses against the secreted transgene product factor IX. Mol. Ther. 1, 225–235 ( 2000).

  52. 52

    Xiao, W. et al. Route of adminstration determines induction of T-cell-independent humoral responses to adeno-associated virus vectors. Mol. Ther. 1, 323–329 ( 2000).

  53. 53

    Wang, L., Takabe, K., Bidlingmaier, S. M., Ill, C. R. & Verma, I. M. Sustained correction of bleeding disorder in hemophilia B mice by gene therapy. Proc. Natl Acad. Sci. USA 96, 3906–3910 ( 1999).

  54. 54

    Scriver, C. R. S., Beaudet, A. L., Sly, W. S. & Valle, D. V. (eds) The Metabolic Basis of Inherited Disease (McGraw–Hill, New York, 1989).

  55. 55

    Cavazzana-Calvo, M. et al. Gene therapy of human severe combined immunodeficiency (SCID)-XI disease. Science 288, 669– 672 (2000).The first definitive example of successful gene therapy, in three children suffering from SCID-XI. The haematopoietic stem cells from the patients were transduced by recombinant retroviruses expressing the γc–subunit, which is common to many interleukin receptors.

  56. 56

    Kohn, D. B. et al. T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates. Nature Med. 4, 775–780 (1998).

  57. 57

    Blaese, R. M. et al. T lymphocyte-directed gene therapy for ADA-SCID: initial trial results after 4 years. Science 270, 475– 480 (1995).

  58. 58

    Bordignon, C. et al. Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients. Science 270, 470–475 (1995).

  59. 59

    Miyoshi, H., Smith, K. A., Mosier, D. E., Verma, I. M. & Torbett, B. E. Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors. Science 283, 682– 686 (1999).

  60. 60

    Guenechea, G. et al. Transduction of human CD34+CD38 bone marrow and cord-derived SCID-repopulating cells with third-generation lentiviral vectors. Mol. Ther. 1, 566– 573 (2000).References 59 and 60 show successful long-term transduction of human haematopoietic stem cells by lentiviral vectors, without the use of agents such as growth factors and cytokines.

  61. 61

    Snyder, R. O. et al. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nature Genet. 16, 270–276 (1997).

  62. 62

    Wang, L., Nichols, T. C., Read, M. S., Bellinger, D. A. & Verma, I. M. Sustained expression of therapeutic level of factor IX in hemophilia B dogs by AAV-mediated gene therapy in liver. Mol. Ther. 1, 154– 158 (2000).

  63. 63

    May, C. et al. Therapeutic haemoglobin synthesis in thalassaemic mice expressing lentivirus-encoded human-globin. Nature 406, 82–86 (2000).

  64. 64

    Cosset, F. L. & Russell, S. J. Targeting retrovirus entry. Gene Ther. 3, 946–956 ( 1996).

  65. 65

    Wickham, T. J. Targeting adenovirus. Gene Ther. 7, 110– 114 (2000).

  66. 66

    Boerger, A. L., Snitkovsky, S. & Young, J. A. Retroviral vectors preloaded with a viral receptor-ligand bridge protein are targeted to specific cell types. Proc. Natl Acad. Sci. USA 96, 9867–9872 (1999).

  67. 67

    Bartlett, J. S., Kleinschmidt, J., Boucher, R. C. & Samulski, R. J. Targeted adeno-associated virus vector transduction of nonpermissive cells mediated by a bispecific F(ab'γ)2 antibody. Nature Biotechnol. 17, 181–186 ( 1999); erratum 17, 393 (1999).

  68. 68

    Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl Acad. Sci. USA 89, 5547–5551 (1992).First description of a regulable gene expression system that uses tetracycline. The transgene is `turned off' in the presence of tetracycline and `turned on' in its absence.

  69. 69

    Urlinger, S. et al. Exploring the sequence space for tetracycline-dependent transcriptional activators: Novel mutations yield expanded range and sensitivity. Proc. Natl Acad. Sci. USA 97, 7963– 7968 (2000).

  70. 70

    Maxwell, I. H., Spitzer, A. L., Long, C. J. & Maxwell, F. Autonomous parvovirus transduction of a gene under control of tissue-specific or inducible promoters. Gene Ther. 3, 28 –36 (1996).

  71. 71

    Kafri, T., Van Praag, H., Gage, F. H. & Verma, I. M. Lentiviral vectors: Regulated gene expression. Mol. Ther. 1, 516–521 (2000).

  72. 72

    Wang, Y., O'Malley, B. W. Jr, Tsai, S. Y. & O'Malley, B. W. A regulatory system for use in gene transfer. Proc. Natl Acad. Sci. USA 91, 8180– 8184 (1994).

  73. 73

    No, D., Yao, T. P. & Evans, R. M. Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc. Natl Acad. Sci. USA 93, 3346–3351 (1996).

  74. 74

    Suhr, S. T., Gil, E. B., Senut, M. C. & Gage, F. H. High level transactivation by a modified Bombyx ecdysone receptor in mammalian cells without exogenous retinoid X receptor. Proc. Natl Acad. Sci. USA 95, 7999–8004 (1998).

  75. 75

    Spencer, D. M., Wandless, T. J., Schreiber, S. L. & Crabtree, G. R. Controlling signal transduction with synthetic ligands. Science 262, 1019–1024 ( 1993).

  76. 76

    Ye, X. et al. Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer. Science 283, 88–91 (1999).

  77. 77

    Miyoshi, H., Takahashi, M., Gage, F. H. & Verma, I. M. Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc. Natl Acad. Sci. USA 16, 10319 –10323 (1997).

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DATABASE LINKS

CD4

OTC

OTC gene

SCID-XI

γC cytokine receptor subunit

ADA

ADA gene

Haemophilia A

Haemophilia B

factor IX

factor IX knockout mice

haemophilic dogs

β-thalassaemia

ecdysone receptors

FURTHER INFORMATION

NIH recombinant DNA advisory committee meeting 8–10 March, 2000

The Institute for Human Gene Therapy

Verma lab homepage

Glossary

LIPOSOMES

Artificial lipid vesicles. Liposomes fuse with the cell membrane to deliver their contents, such as DNA for gene therapy.

EPISOMES

DNA molecules that are maintained in the nucleus without integrating into the chromosomal DNA.

TRANSDUCTION

The introduction of a gene into a target cell by a viral vector.

INTERNAL RIBOSOME ENTRY SITE

A sequence that is inserted between the coding regions for two proteins and allows efficient assembly of the ribosome complex in the middle of a transcript, leading to translation of the second protein.

HAEMATOPOIESIS

The programme of cellular differentiation leading to the formation of blood cells.

KARYOPHILIC

Literally, attracted to the nucleus — a nuclear localization signal in a protein is karyophilic.

TROPISM

The range of cells that can be productively infected by a virus.

CAPSID

The proteinaceous coat surrounding a virus.

RESTENOSIS

Stenosis is the blocking of a blood vessel that can be cleared by mechanical disruption. Restenosis is the recurrence of the blockage caused, for example, by unchecked proliferation and migration of vascular smooth muscle cells.

MEMORY CELLS

Immune cells that are primed, after an initial exposure to an antigen, to make a rapid response to subsequent exposure to the same antigen.

SEROTYPES

Antigenically distinct forms that elicit different antibody responses by the immune system.

DENDRITIC CELLS

These cells present antigen to T cells, and stimulate cell proliferation and the immune response.

TOLERANCE

The lack of an immune response to a specific foreign protein.

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